Coronavirus-like particles comprising functionally deleted genomes

ABSTRACT

The invention relates to the field of coronaviruses and diagnosis, therapeutic use, and vaccines derived therefrom. The invention provides replicative coronaviruses and virus-like particles (VLPs) from which large parts of their genome are (at least functionally) deleted without abolishing their replicative capacities. The deletion preferably results in at least a functional deletion in that the corresponding gene is not or is only partly expressed wherein the resulting gene product is dysfunctional or at least functionally distinct from a corresponding wild-type gene product. One result seen with VLPs provided with deletions as provided herein is that the deleted VLP, albeit capable of replication in vitro and in vivo, are generally well attenuated, in that they do not cause disease in the target host, making them very suitable for therapeutic use, such as a delivery vehicle for genes and other cargo (wherein specific targeting may be provided as well when desired), and for use as a vaccine, being attenuated while carrying important immunogenic determinants that help elicit an immune response.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of PCT International PatentApplication No. PCT/NL/02/00318, filed on May 17, 2002, designating theUnited States of America, and published, in English, as PCTInternational Publication No. WO 02/092827 A2 on Nov. 21, 2002, thecontents of the entirety of which are incorporated herein by thisreference.

TECHNICAL FIELD

The invention relates to the field of coronaviruses and diagnosis,therapeutic use and vaccines derived thereof.

BACKGROUND

Coronavirions have a rather simple structure. They consist of anucleocapsid surrounded by a lipid membrane. The helical nucleocapsid iscomposed of the RNA genome packaged by one type of protein, thenucleocapsid protein N. The viral envelope generally contains 3 membraneproteins: the spike protein (S), the membrane protein (M) and theenvelope protein (E). Some coronaviruses have a fourth protein in theirmembrane, the hemagglutinin-esterase protein (HE). Like all viruses,coronaviruses encode a wide variety of different gene products andproteins.

Most important among these are the proteins responsible for functionsrelated to viral replication and virion structure. However, besidesthese elementary functions, viruses generally specify a diversecollection of proteins, the function of which is often still unknown,but which are known or assumed to be in some way beneficial to thevirus. These proteins may either be essential-operationally defined asbeing required for virus replication in cell culture- or dispensable.Coronaviruses constitute a family of large, positive-sense RNA virusesthat usually cause respiratory and intestinal infections in manydifferent species. Based on antigenic, genetic and structural proteincriteria they have been divided into three distinct groups: Group I, IIand III. Actually, in view of the great differences between the groups,their classification into three different genera is presently beingdiscussed by the responsible ICTV Study Group. The features that allthese viruses have in common are a characteristic set of essential genesencoding replication and structural functions. Interspersed between andflanking these genes, sequences occur that differ profoundly among thegroups and that are, more or less, specific for each group.

Of the elementary genes, the most predominant one occupies abouttwo-thirds of the genome. Located at the 5′ end, this so-called“polymerase gene” encodes two large precursors, the many functionalcleavage products of which are collectively held responsible for RNAreplication and transcription. The other elementary genes specify thebasic structural proteins N, M, E, and S. The nucleocapsid (N) proteinpackages the viral RNA forming the core of the virion. This RNPstructure is surrounded by a lipid envelope in which the membrane (M)protein abundantly occurs constituting a dense matrix. Associated withthe M protein are the small envelope (E) protein and the spike (S)protein, the latter forming the viral peplomers that are involved invirus-cell and cell-cell fusion. The genes for these structural proteinsinvariably occur in the viral genome in the order 5′-S-E-M-N-3′.

In infected cells, the coronavirus nucleocapsids are assembled in thecytoplasm. The nucleocapsids interact with the viral envelope proteinswhich, after their synthesis in the endoplasmic reticulum, accumulate inthe intermediate compartment, a membrane system localized between theendoplasmic reticulum (ER) and the Golgi complex. This membrane systemacts as the budding compartment: the interaction of the nucleocapsidswith the viral envelope proteins leads to the pinching off of virionsthat are then released from the cell by exocytosis.

We have recently demonstrated that the assembly of coronaviral particlesdoes not require the involvement of nucleocapsids. Particles devoid of anucleocapsid are assembled in cells when the viral envelope proteingenes are co-expressed. The minimal requirements for the formation ofvirus-like particles (VLPs) are the M and E protein: the S protein isdispensable but is incorporated if present through its interactions withthe M protein. Biochemical and electron microscopical analysis revealedthat the VLPs are homogeneous in size and have similar dimensions asauthentic corona virions. Clearly, the M and E protein have the capacityto associate in the plane of cellular membranes and induce curvatureleading to the budding of specific “vesicles” which are subsequentlysecreted from the cells. An article describing these results hasappeared in EMBO Journal (vol. 15, pp. 2020-2028, 1996).

In yet another article, coronavirus like particles were shown which werenot devoid of a nucleocapsid, assembly here did not take placeindependent of a nucleocapsid (Bos et al., Virology 218, 52-60, 1996).Furthermore, packaging of RNA was not very efficient. Furthermore,neither of these two publications provides sufficient targeting anddelivery features which would make the VLPs suitable as therapeuticcarrier, for example being equipped with specific targeting informationand/or with a genetic or nongenetic message.

However, coronaviruses do have several distinct theoretical advantagesfor their use as vectors over other viral expression systems (see, also,PCT International Publication WO98/49195): (i) coronaviruses aresingle-stranded RNA viruses that replicate within the cytoplasm withouta DNA intermediary, making unlikely the integration of the virus genomeinto the host cell chromosome; (ii) these viruses have the largest RNAgenome known having, in principle, room for the insertion of largeforeign genes; (iii) since coronaviruses in general infect the mucosalsurfaces, both respiratory and enteric, they may be used to induce astrong secretory immune response; (iv) the tropism of coronaviruses maybe modified by the manipulation of the spike (S) protein allowing theengineering of the tropism and virulence of the vector; and, (v)nonpathogenic coronavirus strains infecting most species of interest areavailable to develop expression systems.

Two types of expression vectors have been developed based on coronavirusgenomes. One requires two components (helper dependent) and the other asingle genome that is modified either by targeted recombination or byengineering a cDNA encoding an infectious RNA. Helper dependentexpression systems, also called minigenomes have been developed usingmembers of the three groups of coronaviruses. Coronavirus derivedminigenomes have a theoretical cloning capacity close to 25 kb, sinceminigenome RNAs of about 3 kb are efficiently amplified and packaged bythe helper virus and the virus genome has about 30 kb. This is, inprinciple, the largest cloning capacity for a vector based on RNA virusgenomes. Reverse genetics has been possible by targeted recombinationbetween a helper virus and either nonreplicative or replicativecoronavirus derived RNAs (9). Targeted recombination has been mediatedby one or two cross-overs. Changes were introduced within the S genethat modified MHV pathogenicity. The gene encoding green fluorescentprotein (GFP) was inserted into MHV between genes S and E by targetedrecombination, resulting in the creation of a vector with the largestknown RNA viral genome (1d). Mutations have also been created bytargeted mutagenesis within the E and the M genes showing the crucialrole of these genes in assembly.

DISCLOSURE OF THE INVENTION

The construction of a full-length genomic cDNA clone could considerablyimprove the genetic manipulation of coronaviruses. The construction ofan infectious TGEV cDNA clone has recently been made possible (1c). Toobtain an infectious cDNA, three strategies have been combined; (i) theconstruction of the full-length cDNA was started from a DI that wasstably and efficiently replicated by the helper virus. Using this DI,the full-length genome was completed and the performance of the enlargedgenome was checked after each step. This approach allowed for theidentification of a cDNA fragment that was toxic to the bacterial host.This finding was used to advantage by reintroducing the toxic fragmentinto the viral cDNA in the last cloning step; (ii) in order to expressthe long coronavirus genome, and to add the 5′ cap, a two-stepamplification system that couples transcription in the nucleus from theCMV promoter, with a second amplification in the cytoplasm, driven bythe viral replicase, was used; and, (iii) to increase viral cDNAstability within bacteria, the cDNA was cloned as a bacterial artificialchromosome (BAC), that produces only one or two plasmid copies per cell.The full-length cDNA was divided into two plasmids because their fusioninto one reduced the stability of the cDNA. One plasmid contained allvirus sequences except for a fragment Cla I to Cla I of about 5 kb thatwas included within a second BAC. A fully functional infectious cDNAclone, leading to a virulent virus able to infect both the enteric andrespiratory tracts, was engineered by inserting the Cla I fragment intothe rest of the TGEV cDNA sequence.

As said, both helper-dependent expression systems, based on twocomponents and single genomes constructed by targeted recombination orby using an infectious cDNA have been developed. The sequences thatregulate transcription have been characterized. Expression of highamounts of heterologous antigens (1 to 8 μg/10⁶ cells) have beenachieved, and the expression levels have been maintained for around 10passages. These expression levels should be sufficient to eliciteprotective immune responses.

Single genome coronavirus vectors have been constructed efficientlyexpressing a foreign gene such as GFP. Thus, a new avenue has beenopened for coronaviruses which have unique properties, such as a longgenome size and enteric tropism, that makes them of high interest asexpression vectors, be it that for vaccine development and gene therapyalso other conditions need be met, notably that host virulence of vectorconstructs, and viability of vector constructs in cell culture need tobe within distinct limitations. One the one hand, the vector may notremain too virulent, but should have at least some attenuated propertiesrendering it useful for in vivo replication as a gene delivery vehicleor vaccine for the host or subject undergoing therapy. On the otherhand, the vector should replicate well in cell-culture, at least whencommercial use is desired.

The invention provides replicative coronaviruses and replicativecoronavirus-like particles (VLPs) from which large parts of their genomeare (at least functionally) deleted and/or are rearranged withoutabolishing their replicative capacities. The deletion is preferablyresulting in at least a functional deletion in that the correspondinggene is not, or is only partly, expressed wherein the resulting geneproduct is absent, dysfunctional or at least functionally distinct froma corresponding wild-type gene product. One striking result seen withVLPs provided with deletions and/or rearrangements as provided herein,is that the deleted or rearranged VLP, albeit capable of replication invitro and in vivo, is in general well attenuated, in that it does notcause disease (or causes much less) in the target host, making it verysuitable for therapeutic use, for example, as a delivery vehicle forgenes and other cargo (wherein specific targeting may be provided aswell when desired), or for use as a vaccine, being attenuated whilecarrying important immunogenic determinants that help elicit an immuneresponse. Such determinants may be derived from a (homologous orheterologous) coronavirus, but may also be derived from other pathogens.In other words, the invention provides the use of a replicative VLP witha partly deleted and/or rearranged genome as a vector. Into the vector,a foreign nucleic acid sequence may be introduced. This foreign genesequence encodes (part of) a protein. It is this protein, or partthereof, encoded by the inserted sequence, which may serve as animmunogen or a “targeting means” (i.e., a ligand capable of binding to areceptor).

In a preferred embodiment, the VLP as provided herein are furthermodified in one of various ways, genomically or in their proteincomposition, thereby exposing at their surface various biological ortarget molecules and/or carrying within the particles molecules withbiological activity which need to be protected or shielded and/orcontaining genomes into which foreign genes or sequences have beenincorporated. One of the major needs in present-day medicine is systemsfor the targeted delivery of therapeutic agents in the body. Byconsequence, the development of carriers that can direct cargo tospecified groups of cells and introduce this cargo into these cells suchthat it can exert its biological activity, is a major challenge inbiomedical research. Tremendous efforts have already been spent in thedevelopment and testing of systems based on liposomes, microspheres,antibodies, etc. for delivery of drugs, genes, peptides and proteins.Though many of these approaches are promising, the actual successes sofar are limited. The genome of a VLP as provided herein has been deletedand/or rearranged to such an extent without abolishing replication thatit serves very well as a delivery vehicle for the cargo. Providing theVLP with cargo or using the VLP as a vector is, e.g., done by insertinga foreign gene sequence that encodes a desired molecule such as a ligandor binding molecule, whether this is an immunogen or receptor bindingmolecule or any other protein or peptide. In a preferred embodiment, thecargo comprises a nucleic acid into which foreign genes or sequences ofinterest have been incorporated. Foreign genes can be expressed by a VLPboth by the additional insertion of such a gene in its genome or byusing the genetic space created by deletion of nonessential genes. Tofurther increase the safety of gene delivery therapy with corona-likeviruses, such as with VLP as provided herein, the invention alsoprovides a method for inhibiting or blocking infection withcoronaviruses in general and with a coronavirus-like particle inparticular, comprising treatment of an organism or cells at risk for aninfection or infected with such a coronavirus or VLP with a so-calledheptad repeat peptide as provided herein. All coronaviruses have hereinbeen found to contain one or two characteristic heptad repeat regions intheir S protein, which are instrumental in coronavirus entry into cells.Peptides derived from the membrane-proximal heptad repeat region (HR2;e.g., for MHV strain A59 the peptide composed of amino acids 1216-1254of the S protein, see also FIG. 20) are particularly potent ininhibiting infection as well as fusion of infected cells withsurrounding ones, as was determined in the detailed description,illustrating the advantages provided. Peptide therapy is not restrictedto situations of gene therapy or delivery vehicle therapy as providedhere, but can also be used in the prevention or treatment of coronaviralinfections in general.

In a further embodiment, the invention provides a replicative VLPaccording to the invention which is also modified at the ectodomainand/or the endodomain of a viral protein. For example, by modifying theectodomain of the spike protein, the VLP is provided with modifiedbiological molecules as targeting means that serve to direct the VLP tointeract with other biological molecules that mirror or can interact,with the target means, such as receptor proteins on cells, be it hormonereceptors, specific immunoglobulinimmunoglobulins on B-cells, MHC andMHC associated molecules present on T-cells and other cells, transferproteins or other receptor molecules known to the person skilled in thefield of cell surface receptors. The targeting means can also beprovided to interact with known binding sites of selected enzymes onproteins or other molecules that serve as substrate for the selectedenzyme.

In another embodiment, replicative VLPs are provided exposing animmunogenic determinant, such as a bacterial toxin or a heterologousviral or bacterial protein comprising a relevant antigenic determinantspecific for a pathogen. This is an example where the VLPs serve asimmunogen or vaccine, here, for example, directed against the bacterialtoxin. B-lymphocytes carrying the correspondingimmunoglobulinimmunoglobulin at their surface are in this case thetarget cells for the VLPs, once recognized by the B-lymphocyte, thiscell(s) will multiply and produce the appropriate antibody.

Preparation of VLPs or coronaviruses with modified spikes can beachieved genetically by modification of the viral genome such that itexpresses the modified S protein in infected cells. Here, we alsoprovide the preparation of coronaviruses containing altered spikes in adifferent way by expressing modified S genes in cells which are inaddition infected with coronavirus. The co-incorporation of the mutantspike provides the virus with new targeting means. In one embodiment,the invention provides a corona-like viral particle (VLP) comprising agenome from which at least a fragment of one of several genes or geneclusters not belonging to the genes specifying the polymerase functions(ORF1a/1b) or the structural proteins N, M, E, and S, are deletedwithout resulting in a total loss of replicative capacities, therebyproviding these VLP with advantageous properties for therapeutic use,for example, as a vector for gene delivery purposes or for use as avaccine delivering a suitable antigen or epitope for eliciting an immuneresponse in the host of interest. In other words, thenonessentialnonessential genes of coronaviruses are not crucial for invitro growth but determine viral virulence. The attenuation acquired bytheir deletion thus provides excellent viral vaccines and therapeuticvectors. Gene delivery or vaccination with a coronavirus can now beachieved with the assuring knowledge that the virus-like particledelivering the gene or antigen of interest is sufficiently attenuated tonot cause specific coronaviral disease. Of course, besides a VLP beingonly deleted in any one or several of the above mentionednonessentialnonessential genes, the invention also provides a VLPprovided with a, preferably functional (in that a substantial peptidefragment of at least 4 of the original amino acids, preferably of atleast 40, is notexpressed), deletion in any of the genes encoding thestructural proteins, in particular, such a deletion in a structuralprotein leads to a VLP with modified spike protein (S) in which part ofthe nucleic acid encoding the spike is deleted and optionally replacedby a foreign gene sequence, thus for example providing the spike withsomething other than the natural ectodomain (or endodomain) of the spikeprotein of the original coronavirus.

The viruses of group I, with the feline infectious peritonitis virus(FIPV) probably as the most complex member, have their typical geneslocated between genes S and E and downstream of the N-gene, i.e.,between N-gene and 3′ UTR (untranslated region). Group II viruses, towhich the mouse hepatitis viruses (MHV) belong, have their particulargenes between the polymerase and S genes and between the genes for the Sand E proteins. One of the encoded proteins characteristic for thisgroup is the hemagglutinin-esterase (HE) protein, which is incorporatedinto virions and of which the hemagglutinating and esterase activitieshave been demonstrated. HE activities are not essential and theirsignificance remains to be elucidated. Finally, also the group IIIviruses, with the prototype coronavirus infectious bronchitis virus(IBV) as the representative, have sequences between the S and E genesbut also between the M and N genes. For some of the group-specificgenes, no expression products could be detected in infected cells, whilefor others (e.g., the HE gene in MHV), naturally occurring viral mutantscarrying deletions in these genes have been observed, the possibilityexists that for some of these genes, not the protein products but othergene products, such as the nucleotide sequences (DNA or RNA) per se, areimportant or essential.

Considering that, based on their genome organization, three groups ofcoronaviruses can be distinguished, the invention provides for group I(FCoV) a recombinant VLP from which preferably a fragment (preferablyresulting in at least a functional deletion in that the correspondinggene is not or is only partly expressed wherein the resulting geneproduct is absent, dysfunctional or at least functionally distinct froma corresponding wild-type gene product) from gene 3a, 3b, 3c, 7a or 7b,or the gene as a whole has been deleted. Such a deleted VLP, albeitcapable of replication in vitro and in vivo, is well attenuated, in thatit does essentially not cause disease in the target host, making it verysuitable for therapeutic use, for example, as a delivery vehicle forgenes and other cargo (wherein specific targeting may be provided aswell when desired), and for use as a vaccine, being attenuated whilecarrying important immunogenic determinants that help elicit an immuneresponse. Such a deleted group I virus, especially a feline coronavirus,such as FIPV, from which a fragment corresponding to gene 3c, and/or 7bis deleted, is in particular immediately useful as a vaccine in that itexpresses relevant antigenic and immunogenic determinants through itsstructural (among others, spike) proteins and is functionally deleted insuch a way that attenuation is achieved, leading to a safe andefficacious vaccine. These live attenuated viruses still induce thefullest spectrum of humoral and cellular immune responses required toprotect against infection and/or disease. Additionally, such a deletedVLP for the prevention of feline disease can be provided withheterologous proteins (or functional fragments thereon, such as(glyco)proteins of feline leukemia virus, feline calicivirus, felineherpes virus, allowing the production of a bivalent or even multivalentvaccine. When desired, other immunologically or therapeuticallyimportant polypeptides, such as cytokines, are incorporated instead oras well.

Furthermore, the invention provides for group II (MHV) a recombinant VLPfrom which preferably a fragment (preferably resulting in at least afunctional deletion) from gene 2a, HE, 4a, 4b, or 5a, or the gene as awhole has been deleted. Such a deleted group II virus, especially anMHV, is provided with advantageous properties for therapeutic use, forexample, especially as a vector for delivery purposes. Such a deletedVLP, albeit capable of replication in vitro and in vivo, is wellattenuated, in that it does essentially not cause disease in the targethost, making it very suitable for therapeutic use, as a delivery vehiclefor genes and other cargo (wherein specific targeting may be provided aswell as when desired), and for use as a vaccine, being attenuated whilecarrying important immunogenic determinants that help elicit an immuneresponse.

For group III (IBV), a recombinant VLP is provided from which preferablya fragment (preferably resulting in at least a functional deletion) fromgene 3a, 3b, 5a or 5b, or the gene as a whole has been deleted. Inanother embodiment, for groups I, II, or III, the invention provides avirus-like particle capable of replication, the particle derived from acoronavirus wherein the genes for the structural proteins do not occurin the order 5′-S-E-M-N-3′. Such a replicative VLP with rearranged geneorder has two important features. One is safety, resulting from the factthat homologous recombination of the VLP genome with that of anaccidental field virus is unlikely to generate viable new progeny. Theother is attenuation due to the shuffling of the genes. Such areplicative VLP with rearranged gene order provides well attenuated VLPsfor vaccine or gene delivery use, and is herein provided bearing thedeletions from the nonessential genes as well. It is shown herein thatchanges in the so-called invariable order of the genes specifying thepolymerase functions (ORF1a/1b) and the structural proteins S, E, M, andN in the coronaviral genome is surprisingly well tolerated, for exampleVLPs with gene order S, M, E, N, or E, S, M, N are easily obtained. Alsohere, foreign genes can be inserted at different positions in the viralgenome, either as an additional gene or replacing deleted nonessentialgenes; these genes are expressed and are stably maintained duringpassage of the virus.

In another embodiment, the invention provides a recombinant VLP wherenucleic acid encoding the S-protein has been modified or at least partlydeleted. The S protein of these viruses is responsible for binding tothe cell receptor and for subsequent fusion of viral and cellularmembrane during entry. These two functions occur in separate regions ofthe molecule: receptor binding in the amino-terminal and fusion in thecarboxy-terminal part. Therefore, by replacing (parts of) the receptorbinding domain by biological molecules with different targetingspecificities, coronaviruses can be directed to interact with a widevariety of target molecules that are, for instance, expressed on thesurface of cells. Doing so without affecting the fusion function of theS-protein, the VLP according to the invention can fuse with or penetrateinto cells not normally injectable by the original virus.

The invention provides virus-like particles (VLPs) derived fromcoronaviruses in which one or more copies of the viral membrane proteinshave been modified so as to contain foreign protein moieties of viral(either coronaviral or noncoronaviral) or nonviral origin, whichmoieties either are replacing part(s) of the VLP membrane proteins orare incorporated within these membrane proteins thereby constituting anintegral part of them. By this, the VLP is provided with novelbiological properties such as new targeting means, or immunologicalinformation, or proteins with specific biological activity containedwithin the virus-like particle, which biological properties areassociated with the VLP next to or in place of the natural spike proteinof the original coronavirus.

In one embodiment, recombinant VLPs with deleted and/or rearrangedgenome are provided in which (a part of) the ectodomain (i.e., the partexposed at the outside of the viral particle) of the spike protein hasbeen replaced by the corresponding domain (or part thereof) of the spikeprotein of another coronavirus. Hereby, the VLP has acquired anothercell substrate specificity wherein the VLP is capable of entering cellsotherwise not accessible or susceptible to the original coronavirus. Ina further embodiment of the invention, VLPs are provided which arecomposed of the mouse hepatitis coronavirus (MHV) M and E proteins andwhich contain chimericchimeric spike molecules consisting of thetransmembrane+carboxy-terminal domain of MHV S but the ectodomain of thespike protein of feline infectious peritonitis coronavirus (FIPV). TheseVLPs can now enter feline cells and deliver MHV-like particles.Particles with these chimericchimeric spikes are produced by makingconstructs of the coronavirus MHV S gene in which the region encodingthe amino-terminal domain is replaced by the corresponding domain ofFIPV. These constructs are inserted into plasmids behind a bacteriophageT7 polymerase promoter. The constructs are then co-transfected withplasmids carrying the MHV M and E genes, both also behind the T7promotor, in OST-7 cells which have been infected with a recombinantvaccina virus expressing the T7 polymerase. The resulting VLPs containthe chimericchimeric MHV/FIPV S protein. In another embodiment of theinvention, the VLP is provided by the methods used as above withectodomains of the spike protein of infectious bronchitis coronavirus(IBV), or the ectodomain (or part thereof) of an envelope protein of anyenveloped virus not belonging to the coronaviruses. For example,MHV-based VLPs are provided by the invention that carry at their surfacethe ectodomain of the pseudorabies virus (PRV) glycoprotein gD insteadof the MHV spike ectodomain or the luminal (i.e., amino-terminal) domain(or part thereof) of any nonviral type I membrane protein. In this way,VLPs are provided that have a cell specificity for chicken cells, or pigcells, or cells reactive with the type I membrane protein.

In yet another embodiment, replicative VLPs are produced withmodifications that are contained within the particles. This is achievedby the incorporation of modified constructs of any of the corona viralproteins S, M, E, and HE. In coronavirus particles, these proteins havetheir carboxy-terminal domain enclosed within the interior of the viralenvelope. Thus, foreign protein sequences incorporated within, appendedto or replacing the carboxy-terminal domain are enclosed as well. Inthis way, VLPs can be provided that contain protein moieties, orfragments thereof, from another virus, or nonviral proteins such ashormones, such as erythropoietin. This allows the production of VLPscontaining a biological active protein or fragments thereof, whichis/are shielded by the viral envelope and can be released and/orretrieved later, when the viral membrane is degraded or fused withanother membrane. This allows the in vitro production in cells, or thein vivo production in secretory glands such as milk glands ofbiologically active substance which are otherwise harmful or toxic tothe producing cells, or which for other reasons need to be produced in ashielded form.

In another embodiment, MHV-based VLPs are provided carrying on theirsurface or inside an enzymatically active molecule like furin, or acytokine, or a hormone receptor, or another viral or nonviralpolypeptide with biological activity. In these examples, VLPs areprovided with (additional) targeting means that serve to direct the VLPto cells otherwise not accessible to the original coronavirus. Theinvention provides recombinantly obtained replicative VLPs which arefurther modified at the ectodomain and/or the ectodomain of any of theviral proteins. By modifying the ectodomain of the spike protein, theVLPs are provided with modified biological molecules as a targetingmeans that serve to direct the VLP to interact with other biologicalmolecules that mirror or can interact with the target means, such asreceptor proteins on cells, be it hormone receptors, specificimmunoglobulins on B-cells, MHC and MHC associated molecules present onT-cells and other cells, transfer proteins or other receptor moleculesknown to the person skilled in the field of cell surface receptors. Thetargeting means can also be provided to interact with known bindingsites of selected enzymes on proteins or other molecules that serve assubstrate for the selected enzyme.

Preparation of VLPs or coronaviruses with modified spikes can beachieved genetically by modification of the viral genome such that itexpresses the modified S protein in infected cells. Here we also providethe preparation of coronaviruses containing altered spikes in adifferent way by expressing modified S genes in cells which are inaddition infected with coronavirus. The co-incorporation of the mutantspike provides the virus with new targeting means. As an example, wedemonstrate the production of MHV particles containing thechimericchimeric MHV/FIPV S protein. The chimeric S gene construct isexpressed in L cells which are subsequently infected with wild-type MHVstrain A59 (MHV-A59) or a mutant thereof. The progeny virus released bythe cells contains the modified S protein. To demonstrate the alteredtargeting, the virus was used to infect feline cells that are naturallynot susceptible to MHV. The cells are now infected as shown byimmunofluorescence and produce normal MHV. As another example, wedemonstrate the production of MHV containing chimeric S proteins inwhich part of the S ectodomain has been replaced by the correspondingpart (i.e., the luminal or amino-terminal domain) of the human CD4molecule, as an example of a nonviral protein. These modifiedcoronaviruses have acquired the property to infect HIV-infected cellsand cells expressing HIV envelope glycoprotein through the specificrecognition of the CD4 and HIV gp120 complex. As a result, theHIV-infected cells will undergo a lytic infection, effectively reducingthe number of HIV-infected cells in the body and thereby reducing theseverity of the disease or even terminating the infection.

As another example, we demonstrate the production of a VLP according tothe invention containing spike molecules of which the amino-terminalpart has been replaced by a single chain-antibody fragment recognizing aspecific cell surface protein that is expressed on cells that cannormally not be infected with the coronavirus laying at the basis of theVLP. The modified virus is able to infect these otherwise refractorycells. This example illustrates the principle that in this way, i.e., byinserting very specific targeting information into the viral spike,coronaviruses can be directed to selected cells or tissues. The singlechain-antibody fragment can, for instance, be selected in phage-displaysystems, or in other clonal selection systems of single-chain antibodyfragments known in the field.

Another aspect of the invention relates to the use of the replicativeVLPs or coronaviruses as gene delivery vehicles. This can be achieved indifferent ways. One way is by incorporating foreign genes or sequencesinto the viral genome such that upon entry of the virus into cells thesegenes are expressed or that the inserted sequences become otherwisebiologically active (as is the case with ribozymes or antisense RNAsgenerated by the virus within the cells). The other way uses VLPs topackage foreign RNA into particles by making use of the coronaviralpackaging signal(s). Incorporating foreign sequences into thecoronaviral genome can be accomplished by genetic manipulation using aninfectious (c-)DNA clone, a full-size DNA copy of the viral genome. Itcan also be achieved by RNA recombination in which case RNA representingpart of the viral genome and containing the foreign sequences isintroduced in infected cells allowing the foreign sequences to beincorporated through homologous recombination. Because coronaviruseswill usually kill the cells they infect, it is important for mostpurposes to attenuate them so that they will not kill the cells withwhich they interact. Attenuation is here accomplished by genomicallyaltering the virus through deletion or rearrangement.

As an example of the invention, attenuation is provided by thepreparation of an MHV mutant from which an essential gene has beendeleted by recombination. A mouse cell line is provided in which the MHVE gene has been chromosomally integrated allowing the E protein to beproduced by the expression of the gene. MHV lacking an E gene has beenproduced in normal mouse cells by recombination using a synthetic RNAcontaining a perfect copy of the MHV genomic 3′-end except for the lackof an intact E gene. The E-defective virus is able to grow only in thecells complementing the defect. The virus produced is attenuated suchthat it can infect other mouse cells, but nonproductively: the lack ofan E protein prevents the assembly of progeny. As an example of theprinciple of incorporating foreign genetic sequences into attenuated ornot-attenuated VLPs or coronaviruses and of their expression is thefollowing, provided by the invention. An MHV derived VLP is providedinto which a reporter gene such as LacZ or green fluorescent protein hasbeen recombined and one in which the chimeric MHV/FIPV S gene has beenincorporated. The expression of the genes is shown by blue orgreen-fluorescent staining of VLP infected cells and by the acquiredability to infect feline cells, respectively. The other way to obtaincoronavirus-based delivery vehicles uses VLPs comprising foreign RNAsequences. Incorporation of foreign RNA sequences into these particlesrequires their packaging into nucleocapsids. Viral RNA-packaging bynucleocapsid (N) protein molecules occurs by the recognition of specificsequences, packaging signal(s) by the N protein. In MHV the packagingsignal includes a 69 nucleotides long region in gene 1B. Foreign(noncoronaviral) RNAs containing the coronavirus packaging signal(s), ordefective coronaviral genomes in which these signal(s) have beenretained but into which foreign sequences have been incorporated, areassembled into VLPs when introduced into cells expressing the N, M and E(±S) genes. The VLP can introduce into a target cell a defined RNA thatmay have one of several functions. An example provided by the inventionis a RNA acting as mRNA and specifying a particular protein such as atoxin or an inducer of apoptosis or an antibody fragment. Anotherexample is an antisense RNA or an RNA with ribozyme activity. For mostpurposes it is essential to acquire multiple copies of the RNA in eachcell to obtain the desired effect. This may not be feasible with VLPswhich will only carry one or a few pseudo-NC. The invention thusprovides the RNAs with amplification signals such that they will bemultiplied in the target cell. To achieve this goal, Semliki Forestvirus (SFV) replication sequences are used as the basis of the RNAconstruct. SFV-derived mRNA further comprising the coronavirusencapsidation sequences and specifying a reporter protein are assembledinto VLPs. The SFV-driven amplification allows synthesis of the reporterprotein in cells; in animals the appearance of antibodies to thereporter protein testifies to the productive delivery of the VLPs'content. The invention also provides a VLP which is an antigen orepitope delivery vehicle meant for the induction of specific immuneresponses, cellular and/or humoral, systemic and/or local, including theinduction and production of specific antibodies against proteins, toachieve protection against infection by pathogens, of viral and nonviralorigin. As an example, the invention provides the induction ofantibodies against the reporter protein derived from SFV-derived mRNAfurther comprising the coronavirus encapsidation sequences andspecifying a reporter protein, as described above. As another example,the induction of antibodies is demonstrated in mice to the FIPV spikeand to PRV gD by immunization with the VLPs, also described hereinabove.Thus, immune responses can be elicited both against proteins which areencoded by the altered genome of the VLP and/or against proteins whichhave been incorporated as targeting means in the VLP, thereby partly orwholly replacing the original spike protein. The examples illustrate theapplicability of the approach for the induction of immune responsesagainst proteins as diverse as, for instance, viral, bacterial,parasitic, cellular and hormonal origins.

The invention also provides VLPs which have fully maintained theoriginal spike protein, but which are altered genomically to attenuatethe VLP and/or to encode nucleotide sequences that need to be deliveredat the cells to which the original coronavirus was targeted. Forexample, in this way, intestinal epithelial cells, or respiratoryepithelial cells, that are normally infected by TGEV, or PRCV,respectively, can now interact with VLPs derived from TGEV or PRCV, orother cell-specific coronaviruses if needed, to express proteinsnormally not expressed by the viruses. In this way, respiratoryepithelial cells of cystic fibrosis patients can, for instance, beinduced to express lung surfactant molecules that are encoded by thealtered genome of the VLP. To further demonstrate the invention, variousexamples are provided in the detailed description which is not limitingthe invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1: A. The top part shows the principle of the RNA recombinationtechnology. Feline cells are infected with fMHV (the MHV-derivativecarrying a chimeric S protein with an ectodomain from the FIPV S proteinallowing the virus to grow in feline cells) and transfected withsynthetic donor RNA that carries, among others, the intact MHV S gene.Proper RNA recombination leads to viruses that have regained the abilityto grow in murine cells by the acquisition of the cognate spikes. Thelower part shows, on the left, the schematic representation of therelevant parts of the plasmid constructs from which donor RNAs weregenerated. On the right, the genomic organization of the recombinantviruses generated with these donor RNAs are depicted.

B. The nucleotide sequences are shown of the new junctions created inthe plasmid constructs (and the resulting viruses), the positions andnumbers of which are indicated in part A (left side). The firstsequences is SEQ ID NO:33. The second sequence is SEQ ID NO:34. Thethird sequence is SEQ ID NO:35.

FIG. 2: RT-PCR analysis of recombinant viruses with genetic deletions.Genomic RNA was isolated from cloned virus, cDNA was prepared by RT withproper primers (1092 and 1127) and PCR was done with primers 1261+990 orwith primers 1173+1260 to analyze the region of genes 4a/b/5a or ofgenes 2a+HE, respectively. The outline of the analyses is shown at theright, the results of the agarose gel analyses of the PCR products isshown at the left. The viruses analyzed are shown above the gel. In theright lane marker DNAs were run.

FIG. 3: Viral RNAs synthesized in infected cells by the different MHVdeletion mutants were analyzed by extraction of total cytoplasmic RNA,metabolically labeled with {³³P]orthophosphate in the presence ofactinomycin D, followed by electrophoresis in 1% agarose gel. Thegenetic make-up of the deletion viruses is shown at the top while thesubgenomic RNA species are designated at the right also according totheir genetic composition.

FIG. 4: One-step growth curves of the recombinant deletion viruses.After high-m.o.i. infection of LR7 cells with the deletion viruses andMHV-WT samples were taken from each culture medium at different timesand the infectivity in these samples was analyzed by titration.

FIG. 5: As in FIG. 1 the construction of viruses with rearranged geneorder is depicted. On the left are the relevant parts of plasmidconstructs: the genome organization of their MHV cDNA sequences and thedesignations of the plasmids. On the right are the respective viruseswith their genome structure and their names.

FIG. 6: One-step growth curves of the recombinant viruses withrearranged genome. After high-m.o.i. infection of LR7 cells with theviruses MHV-Ä2aHE (Δ2aHE), MHV-1bMS, MHV-MSmN, or MHV-WT samples weretaken from each culture medium at different times and the infectivity inthese samples was analyzed by titration.

FIG. 7: A. As in FIG. 1 the construction of viruses with foreign geneinsertions is depicted. At the left the various plasmid (vector)constructs, with their names, are depicted. At the right, the geneticmake-up of viruses obtained with these plasmids by RNA recombination infeline cells are shown together with their names. Below: the primersequences (and numbers) used for the introduction of an intergenicpromoter sequence (IGS) in front of the renilla (RL) and fireflyluciferase (FL) gene. Primer 1286 is SEQ ID NO:16, and Primer 1287 isSEQ ID NO:36.

B. RT-PCR analysis of recombinant viruses carrying the RL gene. cDNA wasprepared using primer 1412 by RT on viral RNA; for each virus twoindependently derived viruses (designated by the extensions A1A and B1A;A2 and B1; A7A and B2D) were analyzed in parallel. Subsequently, PCR wascarried out on the resulting cDNA as well as on the plasmids used togenerate the viruses (see FIG. 7A) with the primer pairs indicated atthe bottom of the figure. For each set, a negative control sample (H₂O)was also included. The agarose gel analysis of the PCR fragmentstogether with DNA markers (flanking lanes) are shown and the sizes ofthe products are indicated at the right.

FIG. 8: One-step growth curves of the recombinant viruses carryingforeign genes. After high-m.o.i. infection of LR7 cells with viruses,samples were taken from each culture medium at different times and theinfectivity in these samples was analyzed by titration.

A. Growth curves of different viruses having the renilla luciferase geneas compared to MHV-WT.

B. The growth of two independently obtained clones of MHV-EFLM viruscarrying the firefly luciferase gene is shown as compared to MHV-WT.

FIG. 9: Expression of luciferase by recombinant viruses. LR7 cells wereinfected with viruses expressing the renilla (A) or firefly (B)luciferase and with MHV-WT, and the luciferase activity generated in thecells was monitored over time. In B the two independently obtainedclones of MHV-EFLM virus (see FIG. 8B) are compared to MHV-WT.

FIG. 10: Stability of viruses carrying foreign genes. The recombinantviruses MHV-ERLM and MHV-EFLM (two independently obtained clones in eachcase) were passaged 8 times, over LR7 cells at low m.o.i. After eachpassage, the viral infectivity (TCID50) in the harvested culture mediumwas determined. The collection of viruses thus obtained was inoculatedin parallel into LR7 cells at m.o.i. of 5 and the luciferase expressionin the cells at 8 hours post-infection was quantified. Results areplotted as a function of passage number.

FIG. 11: Inhibition of MHV infection by the HR2 peptide. LR7 cells wereinoculated with the virus MHV-EFLM in the presence of differentconcentrations of HR2 peptide or, as a control, a peptide correspondingto amino acids 1003-1048 of the viral S protein. After one hour ofinoculation, cells were washed and incubated further in the absence ofpeptide. To evaluate the success of the infection, cells were analyzedat 4 hours post-infection for luciferase expression. In the figureluciferase activity is plotted against the different peptideconcentrations used.

FIG. 12: Inhibition of cell-cell fusion by HR2 peptide. Afterinoculation of parallel cultures of LR7, cells with MHV-A59 cells wereoverlaid with agar medium containing different concentrations of HR2peptide and plaques were counted the following day.

FIG. 13: A. The genetic structure of the primary plasmid vector pBRDI1as compared to that of the parental virus from which it was derived. Thetop part shows the genome organization of FIPV strain 79-1146. Thedotted parts (i.e., the very 5′-most 702 bases and the 3′-derived 9,262bases) were assembled into the cDNA construct of pBRDI1; in this plasmidthe viral cDNA is preceded by a phage T7 promoter sequence and theinsert is flanked by XhoI (5′) and NotI (3′) restriction sites.

B. Plasmid pBRDI2 is a derivative of pBRDI1 in which the FIPV S gene hasbeen replaced by a chimeric S gene (designated mS) which encodes theMHV-A59 S ectodomain while having retained the sequences for the FIPV Sprotein transmembrane and endodomain. Plasmid pBRDI2 was obtained bysubstituting in pBRDI1 the SacI-AflII segment by the correspondingfragment of pTMFS1. The latter plasmid is a derivative of plasmid pTMFS(12) which contains the chimeric gene sequence and into which aSacI-StuI fragment was cloned to extend the MHV S sequence at its 5′ endwith sequences corresponding to the FIPV pol1B 3′ end.

FIG. 14: Sequence details of the pBRDI constructs.

A. The nucleotide sequence of pBRDI1 and pBRDI2 around the very 5′ endof the FIPV genome sequence: the XhoI site and the phage T7 sequence arefollowed by a G-triplet and subsequently by the FIPV sequence. Theentire sequence is SEQ ID NO:37.

B. The sequence at the pol1A/pol1B junction. The codon sequence is SEQID NO:38. The amino acid sequence is SEQ ID NO:39.

C. The nucleotide sequence at the very 3′ end of the cDNA construct. The3′ untranslated region (3′ UTR) is followed by a stretch of adeninenucleotides and a NotI sequence. The entire sequence is SEQ ID NO:40.

D. Nucleotide sequence at the FIPV pol1B-MHV S transition in pTMFS1 andpBRDI2. The codon sequence is SEQ ID NO:41. The top amino acid sequenceis SEQ ID NO:42, and the bottom amino acid sequence is SEQ ID NO:43.

FIG. 15: Schematic picture of the generation of mFIPV by therecombination of FIPV genomic RNA and pBRDI2 derived synthetic RNA infeline cells. The genetic organization in each RNA is indicated as wellas the selection for recombinant (i.e., chimeric mS containing) virus bygrowth on murine cells.

FIG. 16: Analysis of the mFIPV structural proteins. Murine LR7 cellswere infected with mFIPV and, for comparison, with MHV-A59; feline FCWFcells were infected with FIPV strain 79-1146. Infected cells werelabeled with ³⁵S-amino acids from 5-7 hours post-infection cell lysateswere prepared and immunoprecipitations were carried out with differentantibodies: polyclonal antibodies against FIPV (lanes 1, 6, and 10) andMHV (lanes 3, 4, 8 and 12) and monoclonal antibodies against the felineS protein (S_(f); lanes 2, 7, and 11) and the murine S protein (S_(m);lanes 5, 9, and 13). The proteins were analyzed by electrophoresis inSDS-12% polyacrylamide gel. The position in the gel of the MHV and FIPVproteins are indicated at the left and right side, respectively. ThemFIPV S protein is precipitated by the MHV S specific sera, not by thoseprecipitating the FIPV S protein, and its migration in gel is similar tothat of the MHV S protein.

FIG. 17: One-step growth curves of mFIPV as compared to MHV. Afterhigh-m.o.i. infection of LR7 cells with the two viruses, samples weretaken from each culture medium at different times and the infectivity inthese samples was analyzed by titration.

FIG. 18: Generation of FIPV deletion mutants. At the top the principleof the method is shown: recombination of the mFIPV RNA with syntheticpBRDI derived donor RNA carrying the intact FIPV S gene. Below this aredepicted the genetic make-up of the constructed pBRDI1 plasmids (left)and of the generated viruses (right). Arrows indicate the position (andnumber) of the primers used, open triangles indicate deletions. At theright, numbers of base pairs (bp) indicate the sizes of PCR fragmentspredicted to be obtained with the indicated primer pairs.

FIG. 19: Genetic analysis of the recombinant FIPV deletion viruses.Genomic RNA was isolated from the deletion viruses as well as fromrecombinant wild-type FIPV. RT and PCR reactions were carried out usingthe primers 1 and 9 for the analysis of the genes 3ABC region (panel A)and primers 10 and 11 for the genes 7AB (panel B). The positions of theDNA fragments in the agarose gels are indicated alongside the geltogether with their predicted size (c.f. FIG. 18, right). The viral RNAsanalyzed in the different lanes are indicated at the right.

FIG. 20: Heptad repeat (HR) regions and their amino acid sequences incoronavirus spike proteins. In the HR1 region, MHV is SEQ ID NO:44,HCV-OC43 is SEQ ID NO:45, HCV-229E is SEQ ID NO:46, FIPV is SEQ IDNO:47, and IBV is SEQ ID NO:48. Peptide HR1 is SEQ ID NO:49. PeptideHR1a is SEQ ID NO:50. Peptide HR1b is SEQ ID NO:51. Peptide HR1c is SEQID NO:52. In the HR2 region, MHV is SEQ ID NO:53, HCV-OC43 is SEQ IDNO:54, HCV-229E is SEQ ID NO:55, FIPV is SEQ ID NO:56, and IBV is SEQ IDNO:57. Peptide HR2 is SEQ ID NO:58.

A. Schematic representation of the coronavirus MHV-A59 spike structure.The spike (S) glycoprotein contains an N-terminal signal sequence (SS)and a transmembrane domain (TM) close to its C-terminus. S isproteolytically cleaved (arrow) in an S1 and S2 subunit, which arenoncovalently linked. S2 contains two conserved heptad repeat regions,HR1 and HR2, as indicated.

B. Sequence alignment of HR1 and HR2 domains of MHV-A59, HCV-OC43 (humancoronavirus strain OC43), HCV-229E (human coronavirus strain 229E), FIPVand IBV (infectious bronchitis virus strain Beaudette). The alignmentshows a remarkable insertion of exactly 2 heptad repeats (14 aa) in bothHR1 and HR2 of HCV-229E and FIPV, which is present in S proteins of allgroup I viruses. The predicted hydrophobic heptad repeat a and dresidues are indicated above the sequence. The frame shift of predictedheptad repeats in HR1 is caused by a stutter. Asteriks denote conservedresidues. The amino acid sequences of the peptides HR1, HR1a, HR1b, HR1cand HR2 used in this study are presented in italics below thealignments. N-terminal residues derived from proteolytic cleavage siteof the GST-fusion protein are between brackets.

FIG. 21=Table 1: The sequences are shown of the junctions that weregenerated in the plasmids depicted in FIG. 5 and in the viruses obtainedwith these plasmids. The numbers correspond to the numbering of thejunctions as indicated by arrowheads in the plasmids (FIG. 5, left). Thefirst sequence is SEQ ID NO:59. The second sequence is SEQ ID NO:60. Thethird sequence is SEQ ID NO:61. The fourth sequence is SEQ ID NO:62. Thefifth sequence is SEQ ID NO:63. The sixth sequence is SEQ ID NO:64. Theseventh sequence is SEQ ID NO:65. The eighth sequence is SEQ ID NO:66.The ninth sequence is SEQ ID NO:67.

FIG. 22=Table 2: Primers used for splicing overlap extension (SOE)-PCRand for RT-PCR in the construction and analysis of recombinant FIPVs.For their position on the FIPV genome: see FIG. 18, in which theirnumbers and sense are indicated by arrows. The numbering of the primersrefers to that in the pBRDI1 sequence. Primer 1 is SEQ ID NO:68. Primer2 is SEQ ID NO:69. Primer 3 is SEQ ID NO:70. Primer 4 is SEQ ID NO:71.Primer 2 is SEQ ID NO:72. Primer 6 is SEQ ID NO:73. Primer 7 is SEQ IDNO:74. Primer 8 is SEQ ID NO:75. Primer 9 is SEQ ID NO:76. Primer 10 isSEQ ID NO:77. Primer 11 is SEQ ID NO:78.

FIG. 23=Addendum 1: Nucleotide sequence of plasmid pBRDI1 (SEQ IDNO:79). The sequence starts with the XhoI site (ctcgag) followed by thephage T7 polymerase promoter sequence and the triple G sequence, afterwhich it proceeds with the 5′ FIPV cDNA sequence.

FIG. 24=Addendum 2: Nucleotide sequence of plasmid pBRDI2 (SEQ IDNO:80). The sequence starts with the XhoI site (ctcgag) followed by thephage T7 polymerase promoter sequence and the triple G sequence, afterwhich it proceeds with the 5′ FIPV cDNA sequence.

FIG. 25: Survival of C57B1/6 mice infected with MHV recombinants.Four-week-old mice were inoculated intracranially with various dilutionsof recombinant wild type and deletion viruses (n=5 per virus) andsurvival was monitored. The data for mice infected with 2.5×10⁵ PFU areshown. While the animals infected with MHV-WT had all succumbed by day 7post-infection, all mice inoculated with the deletion mutant virusessurvived until 21 days post-infection.

FIG. 26A: Infected 17Cl1 cells were metabolically labeled with[³³P]orthophosphate in the presence of actinomycin D essentially asdescribed (4, 10). Samples of total cytoplasmic RNA, purified usingUltraspec reagent (Biotecx), were denatured with formaldehyde andformamide, separated by electrophoresis through 1% agarose containingformaldehyde, and visualized by fluorography. The different RNA speciesare indicated by numbers corresponding with Table 4 (=FIG. 42).

FIG. 26B: Mice inoculated intraperitoneally with 1×10⁶TCID⁵⁰ of each MHVrecombinant were euthanized at day 4 post-infection and the viralreplication in the liver was determined by quantitative plaque assays.

FIG. 27: RT-PCR analysis of recombinant viruses MHV-EFLM andMHV-minFL.cDNA was prepared using primer 1475 by RT on viral RNA; foreach virus two independently derived viruses (designated by theextensions A1A and B1A, A6F and B4A) were analyzed in parallel.Subsequently, PCR was carried out on the resulting cDNA as well as onthe plasmids used to generate the viruses (see, FIG. 7A) with the primerpair 935 and 1474. The agarose gel analysis of the PCR fragmentstogether with a DNA marker is shown.

FIG. 28: RT-PCR analysis of recombinant viruses MHV-EFLM and MHVminFL.cDNA was prepared using primer 1412 by RT on viral RNA; for each virustwo independently derived viruses (designated by the extensions A2E andB4D) were analyzed in parallel. Subsequently, PCR was carried out on theresulting cDNA as well as on the plasmids used to generate the viruses(see, FIG. 7A) with the primer pairs indicated at the bottom of thefigure. The agarose gel analysis of the PCR fragments together with aDNA marker is shown.

FIGS. 29-31: RNA synthesis by recombinant MHVs. The genomes of therecombinant viruses are depicted at the top, their observed RNAexpression patterns are shown below. Infected 17Cl1 cells weremetabolically labeled with [³³P]orthophosphate in the presence ofactinomycin D essentially as described (4, 10). Samples of totalcytoplasmic RNA, purified using Ultraspec reagent (Biotecx), weredenatured with formaldehyde and formamide, separated by electrophoresisthrough 1% agarose containing formaldehyde, and visualized byfluorography. Note that the subgenomic RNA species in this figure aredesignated by their composition, rather than as RNA2 through RNA7, sincethe numerical designations would be ambiguous for the mutants.

FIG. 29: MHV-WT, MHV-ERLM and MHV-EFLM

FIG. 30: MHV-EFLM and MHV-minFL

FIG. 31: MHV-MRLN, MHV-ERLM, MHV-2aRLS, MHV-MSmNRL, and MHV-MSmN

FIG. 32: In vitro replication of the recombinant viruses carryingforeign genes. After high-m.o.i. infection of LR7 cells with viruses(top: MHV-MRLN, MHV-ERLM, MHV-2aRLS; middle: MHV-EFLM, MHV-minFL;bottom, MHV-ERLM, MHV-MSmNRL), samples were taken from each culturemedium at 9 hours post-infection and the infectivity in these sampleswas analyzed by titration.

FIG. 33: Expression of luciferase by recombinant viruses. Intracellularexpression of firefly and renilla luciferase of several recombinantviruses was determined according to the manufacturer's instructions(Promega) at 9 hours post-infection. Top: MHV-EFLM and MHV-minFL;bottom: MHV-ERLM and MHV-MSmNRL.

FIG. 34: Expression of firefly luciferase in vivo. Eight-week-old,MHV-negative, female BALB/c mice were used in the experiment. Mice wereinoculated intranasally with MHV-EFLM. Four animals per dose (10⁶TCID⁵⁰) were used. Mice were sacrificed and the livers and brains wereremoved at day 4 post-infection. Organs were quick-frozen in liquidnitrogen and homogenized in Cell Culture Lysis Reagent provided with theLuciferase Assay System (Promega). FL activity was measured according tothe manufacturer's instructions using a luminometer (Lumac BiocounterM2500).

FIG. 35: Survival rates following inoculation of cats with variousdeletion mutants: FIPW 79-1146; r-wtFIPV; FIPVΔ3abc; FIPVΔ7a; andFIPVΔ3abc+7ab (100 pfu).

FIG. 36: FIPV neutralizing antibody titers raised at different timepoints after infection of cats with FIPV deletion variants.

FIG. 37: Survival rates following challenge of cats with FIPV 79-1146(100 pfu). Cats (N=4) not previously vaccinated (□); cats (N=5)previously vaccinated with FIPVΔ3abc (♦); cats (N=5) previouslyvaccinated with FIPVΔ7ab (□); and cats (N=5) previously vaccinated withFIPVΔ3abc+7ab (□).

FIG. 38: One-step growth curves of the recombinant FIPV viruses carryingforeign genes. After high-m.o.i. infection of FCWF cells with viruses,samples were taken from each culture medium at different times and theinfectivity in these samples was analyzed by titration. Recombinant nr.1 (□) and nr. 9 (♦).

FIG. 39: Expression of luciferase by recombinant viruses. FCWF cellswere infected with viruses expressing the renilla luciferase (nr. 1□;and nr. 9♦) and with wt-rFIPV (□), and the luciferase activity generatedin the cells was monitored over time.

FIG. 40: One-step growth curves of the recombinant FIPV viruses withdeletion of nonessential genes. After high-m.o.i. infection of FCWFcells with viruses, samples were taken from each culture medium atdifferent times and the infectivity in these samples was analyzed bytitration.

FIG. 41=Table 3 Quantifications of RNA synthesis by recombinant MHVscarrying deletions of nonessential genes.

FIG. 42=Table 4 Quantifications of RNA synthesis by recombinant MHVswith rearranged genomes.

FIG. 43=Table 5 Scoring table for clinical signs following vaccinationand challenge.

FIG. 44=Table 6 Total clinical score following initial vaccination withdifferent mutants of FIPV79-1146.

FIG. 45=Table 7 Total clinical score following challenge with FIPV79-1146.

FIG. 46A: Replication of the recombinant virus carrying 2 foreign genes.After high-m.o.i. infection of LR7 cells with viruses (MHV-RLFL,MHV-2aRLS, and MHV-EFLM) samples were taken from each culture medium at9 hours post-infection and the infectivity in these samples was analyzedby titration.

FIG. 46B: Expression of luciferase by recombinant viruses. Intracellularexpression of firefly and renilla luciferase of several recombinantviruses was determined according to the manufacturer's instructions(Promega) at 9 hours post-infection (MHV-RLFL, MHV-2aRLS, and MHV-EFLM).

Experimental data related to the patent:

I. Mouse Hepatitis Virus (MHV), strain A59 (MHV-A59)

1. Generation of live attenuated viruses

-   -   a. Construction of recombinant MHVs lacking genes    -   b. Confirmation of the recombinant genotypes    -   c. RNA synthesis by MHV deletion mutants    -   d. Tissue culture growth phenotype    -   e. Virulence of recombinant viruses in mice

2. Generation of recombinant viruses with rearranged gene order

-   -   a. Construction of recombinant viruses with rearranged gene        order    -   b. RNA synthesis by MHV mutants with rearranged gene order    -   c. Tissue culture growth phenotype    -   d. Replication of recombinant viruses in mice

3. Generation of recombinant viruses expressing foreign genes

-   -   a. Construction of recombinant viruses carrying reporter genes    -   b. RNA synthesis by recombinant viruses    -   c. Replication of viruses expressing renilla or firefly        luciferase    -   d. Expression of renilla and firefly luciferase in cell culture    -   e. Maintenance of foreign genes during viral passage    -   f. Expression of firefly luciferase in mice    -   g. Generation of MHV expressing two foreign genes from one        genome    -   h. Generation of MHV expressing a chimeric Spike-GFP gene

4. Inhibition of infection and of cell fusion by spike protein derivedpeptide

II. Feline Infectious Peritonitis Virus (FIPV), strain 79-1146

1. Generation of mFIPV, a feline coronavirus growing on murine cells

-   -   a. Construction of a synthetic RNA transcription vector    -   b. Generation of mFIPV by RNA recombination    -   c. mFIPV protein analysis    -   d. mFIPV growth characteristics

2. Generation of live attenuated FIPV vaccine by gene deletions

-   -   a. Construction of synthetic RNA transcription vectors    -   b. Generation of recombinant FIPVs lacking genes    -   c. Genetic analysis of recombinant FIPVs lacking genes    -   d. Growth characteristics of FIPVs lacking genes    -   e. Virulence of recombinant viruses in cats    -   f. Immune-response induced by recombinant viruses    -   g. FIPV deletion viruses serve as attenuated, live vaccines

3. Insertion and expression of foreign genes

-   -   a. Construction of recombinant viruses carrying reporter genes    -   b. One-step growth of viruses    -   c. Expression of renilla luciferase

4. Generation of multivalent FIPV-based vaccines

-   -   a. Construction of a multivalent Feline leukemia virus (FeLV)        vaccine based on FIPV vector    -   b. Construction of a multivalent Feline immunodeficiency virus        (FIV) vaccine based on a FIPV vector    -   c. Construction of a multivalent Feline calicivirus (FCV)        vaccine based on a FIPV vector    -   d. Construction of a multivalent Feline panleucopenia virus        (FPV) vaccine based on a FIPV vector    -   e. Construction of a multivalent Feline herpes virus (FHV)        vaccine based on an FIPV vector    -   f. Construction of a multivalent FIPV serotype I and II vaccine        based on an FIPV serotype II vector

5. Generation of recombinant viruses with rearranged gene order

6. Generation of FIPV based vaccines against canine pathogens

-   -   a. Generation of FIPV based vaccine against canine distemper    -   b. Generation of FIPV based vaccine against canine parvo disease    -   c. Generation of FIPV based vaccine against infectious canine        hepatitis    -   d. Generation of FIPV based vaccine against hemorrhagic disease        of pups        III. Transmissible gastro-enteritis virus (TGEV)

1. Generation of a live attenuated vaccine against TGEV

2. Generation of multivalent TGEV-based vaccines

-   -   a. Construction of a multivalent Porcine Parvovirus (PPV)        vaccine based on a TGEV vector    -   b. Construction of a multivalent swine influenza virus vaccine        based on a TGEV vector    -   c. Construction of a multivalent African swine fever virus        vaccine based on a TGEV vector    -   d. Construction of a multivalent Porcine circovirus type 2        vaccine based on a TGEV vector    -   e. Construction of a multivalent Porcine respiratory and        reproductive syndrome virus vaccine based on a TGEV vector

3. Generation of recombinant viruses with rearranged gene order

IV. Avian Infectious Bronchitis Virus (IBV)

1. Generation of a live vaccine based on attenuated IBV

2. Generation of multivalent IBV-based vaccines

-   -   aI. Construction of a multivalent vaccine based on an IBV vector        that protects against more than one IBV serotype.    -   aII. Construction of a multivalent vaccine based on an IBV        vector that protects against Newcastle Disease.    -   b. Construction of a multivalent vaccine based on an IBV vector        that protects against Avian Influenza.    -   c. Construction of a multivalent vaccine based on an IBV vector        that protects against Chicken Anemia Virus (CAV) disease.    -   d. Construction of a multivalent vaccine based on an IBV vector        that protects against Avian reovirus disease.    -   e. Construction of a multivalent vaccine based on an IBV vector        that protects against Infectious Bursal Disease.    -   f. Construction of a multivalent vaccine based on an IBV vector        that protects against Marek's disease.    -   g. Construction of a multivalent vaccine based on an IBV vector        that protects against Infectious laryngotracheitis.

3. Generation of recombinant viruses with rearranged gene order

V. Human Coronavirus (HCoV) strain 229E (HCoV-229E)

1. Generation of a live vaccine based on attenuated HCoV-229E

2. Generation of a live attenuated vaccine against HCoV strain OC43

3. Generation of multivalent HCoV-based vaccines

-   -   a. Construction of a multivalent vaccine based on an HCoV vector        that protects against Respiratory Syncytial Virus (RSV)    -   b. Construction of a multivalent vaccine based on an HCoV vector        that protects against rotavirus    -   c. Construction of a multivalent vaccine based on an HCoV vector        that protects against Norwalk-like viruses    -   d. Construction of a multivalent vaccine based on an HCoV vector        that protects against influenza virus

4. Generation of recombinant viruses with rearranged gene order

DETAILED DESCRIPTION OF THE INVENTION

I. Mouse Hepatitis Virus (MHV), Strain A59 (MHV-A59):

I.1 Generation of Live Attenuated Viruses:

General aim: establish whether deletion from the coronaviral (i.e.MHV-A59) genome of genes or gene clusters not belonging to the genesspecifying the polymerase functions (ORF1a/1b) or the structuralproteins N, M, E, and S, is tolerated and yields viable viruses even ifthese gene sequences are removed altogether; establish whether suchdeletions have an attenuating effect on the virus when inoculated intomice.

I.1.a. Construction of Recombinant MHVs Lacking Genes:

Specific aim: generate MHV-A59 deletion mutants lacking genes 2a+HE(MHV-□Δ2aHE) genes 4a+4b+5a (MHV-□Δ45a), and genes 2a+HE+4a+4b+5a(MHV-min).

Approach: targeted RNA recombination (3, 9, 10) using fMHV, the MHV-A59derivative infecting feline (FCWF) cells not murine (LR7) cells (7), andsynthetic donor RNAs carrying the intended deletions (FIG. 1A, top).

Procedure: Transcription vectors for the production of synthetic donorRNAs were constructed from the plasmid pMH54 (7), which encodes arun-off transcript consisting of the 5′ end of the MHV-A59 genome (467nt) fused to codon 28 of the HE gene and running to the 3′ end of thegenome (FIG. 1). Plasmid pMH54 was used to reconstruct the recombinantWT-MHV, an fMHV derivative again infecting murine cells. Transcriptionvector pXH□Δ45a lacks the ORFs 4a, 4b, and 5a. For the construction ofthis plasmid, a PCR product was obtained from plasmid pB59 (2) by usingprimer 1089 (5′-ACCTGCAGGACTAATCTAAACTTTATTCTTTTTAGGGCCACGA-3) (SEQ IDNO: 1), which encodes a PstI/Sse8387I restriction site, an intergenicsequence (IGS) and which is complementary to the sequence just upstreamof the E or 5b gene, and primer 1092 (5′-CCTTAAGGAATTGAACTGC-3′) (SEQ IDNO:2) which is complementary to the 5′ end of the M protein codingregion. The PCR product was cloned into pGEM-T Easy (Promega) accordingto the manufacturer's instructions, yielding pXH0803. The PCR productwas subsequently excised with PstI and EcoRV and cloned into pMH54treated with Sse8387I and EcoRV, resulting in pXH□Δ45a. Transcriptionvector pXH□Δ2aHE lacks ORFs 2a and HE and contains approximately 1200 bpof the 3′ end of the polymerase gene fused to the S gene. To constructthis plasmid a PCR product was obtained by splicing overlap extensionPCR. One PCR product was obtained from plasmid p96 (1) using primer 1128(5′-ACGGTCCGACTGCGCGCTTGAACACGTTG-3′) (SEQ ID NO:3) which encodes aRsrII restriction site and is complementary to the region 1200 bpupstream of the ORF1b stop codon, and primer 1130(5′-CATGCAAGCTTTATTTGACATTTACTAGGCT-3′) (SEQ ID NO:4) which iscomplementary to the 3′ end of the polymerase coding region and the IGSregion upstream of the S gene. The other PCR product was obtained frompMH54 using primer 1129 (5′-GTCAAATAAAGCTTGCATGAGGCATAATCTAAAC-3) (SEQID NO:5) which is complementary to primer 1130, and primer 1127(5′-CCAGTAAGCAATAATGTGG-3) (SEQ ID NO:6) which is complementary to the5′ end of the S gene. The PCR products were purified and mixed and thenamplified with primers 1128 and 1127. The PCR product obtained in thesecond round of PCR was cloned into pGEM-T Easy, yielding pXH1802. ThePCR product was excised with RsrII and AvrII and cloned into pMH54treated with the same enzymes, resulting in pXHΔ□2aHE. Transcriptionvector pXHmin has the ORF1b 3′ end fused to the S gene and the deletionof ORF4a, 4b and 5a. This vector was constructed by cloning the fragmentexcised with PstI and EcoRV from pXH0803 into pXH□2aHE treated withSse8387I and EcoRV. The composition of all PCR-generated segments wasconfirmed by DNA sequencing.

To generate the deletion mutant viruses, donor RNAs were transcribedfrom the (PacI-linearized) pMH54-derived plasmids and transfected byelectroporation into feline FCWF cells that had been infected with fMHV.These infected and transfected cells were then plated onto a monolayerof mouse LR7 (7) cells. After 24 hours of incubation at 37° C., progenyviruses were harvested by taking off the cell culture supernatant andcandidate recombinants were selected by two rounds of plaquepurification on LR7 cells.

Result: With each of the synthetic donor RNAs used, clear plaques wereobtained.

Conclusion: Recombinant viruses had been obtained that had regained theability to grow on murine cells.

I.1.b. Confirmation of the Recombinant Genotypes:

Aim: Confirming by RT-PCR, the genetic make-up of the recombinantviruses obtained.

Procedure and Results: Cloned recombinant viruses, one from eachrecombination experiment, were produced on LR7 cells, viral RNA wasisolated and RT-PCR was done using standard methods on genomic RNA asshown in FIG. 2. To confirm the deletion of the ORFs 4 and 5a, the RTstep was performed with primer 1092 (5′-CCTTAAGGAATTGAACTGC-3′) (SEQ IDNO:2), which is complementary to the 5′ end of the M gene, while the PCRwas performed with primer 1261 (5′-GCTGCTTACTCCTATCATAC-3′) (SEQ IDNO:7) and primer 990 (5′-CCTGATTTATCTCTCGATTTC-3) (SEQ ID NO:8) whichare complementary with the 3′ end of the E and S gene, respectively. Inthe case of the recombinant MHV-WT, an RT-PCR product corresponding insize with the expected 1328 bp was observed (FIG. 2, top). As expected,a PCR product of the same length was observed for MHV-Δ□2aHE. Incontrast, both for MHV-□Δ45a and MHV-min much smaller RT-PCR productswere detected. The smaller size of the RT-PCR products corresponded withthe deletion of 736 bp. The deletion of ORFs 2a and HE was analyzed in asimilar way. The RT step was performed with primer 1127(5′-CCAGTAAGCAATAATGTGG-3) (SEQ ID NO:6) which is complementary to the5′ end of the S gene. The PCR was performed with primer 1173(5′-GACTTAGTCCTCTCCTTGA-3′) (SEQ ID NO:9) and primer 1260(5′-CTTCAACGGTCTCAGTGC-3) (SEQ ID NO:10), which are complementary to the3′ end of the 1b gene and the 5′ end of the S gene, respectively. Bothfor MHV-WT and MHV-□Δ45a PCR products were detected, which were muchbigger than the PCR products detected for MHV-Δ2aHE and MHV-min (FIG. 2,bottom). The difference in size corresponded with the deletion of 2164bp. Finally, the newly generated junctions, present in the genomes ofthe deletion mutant viruses (FIGS. 1A [triangles] and 1B [sequence]),were analyzed by sequencing of the RT-PCR products. To this end the PCRproducts were cloned into the pGEM-T easy vector (Promega). Thesequences obtained were in perfect agreement with the predictions.

Conclusion: The constructed viral mutants had the intended geneticdeletions.

I.1.c. RNA Synthesis by MHV Deletion Mutants:

Aim: Confirming the patterns of RNAs synthesized in cells infected bythe mutant viruses.

Procedure: Infected 17Cl1 cells were metabolically labeled with[³³P]orthophosphate in the presence of actinomycin D essentially asdescribed (4, 10). Samples of total cytoplasmic RNA, purified usingUltraspec reagent (Biotecx), were denatured with formaldehyde andformamide, separated by electrophoresis through 1% agarose containingformaldehyde, and visualized by fluorography (FIG. 3; note that thesubgenomic RNA species in this figure are designated by theircomposition, rather than as RNA2 through RNA7, since the numericaldesignations would be ambiguous for the deletion mutants).

Result: For the recombinant MHV-WT, the RNA pattern and the relativemolar amounts of the six subgenomic (sg) RNA species and the genomic (g)RNA were very similar to those reported previously for MHV(10)(4)(5)(8), with one notable exception. The 4-5a/E-M-N sgRNA, whichis usually denoted RNA4, was far more abundant than previously observedfor this species in wild-type MHV (10)(4)(5)(8). This was presumably dueto the three nucleotide changes at positions 13, 15, and 18 upstream ofthe consensus transcription regulatory signal, (5′-AAUCUAAAC3-′) (SEQ IDNO:11) that precedes gene 4 (FIG. 1) which were introduced into thetranscription vector pMH54 to create the Sse8387I site downstream of theS gene (7). For the deletion mutants, all variant sgRNAs had mobilitiesthat corresponded to their predicted sizes (FIG. 3 and Table 3), and noprominent extra species were observed. The relative molar amounts of themutant sgRNA species were quite similar to those of their wild-typecounterparts originating from the corresponding transcription regulatorysignals.

Conclusion: The results confirm the genotypes of the recombinant virusesand demonstrate their expected phenotypes at the RNA level.

I.1.d. Tissue Culture Growth Phenotype:

Aim: Comparing the in vitro growth phenotypes.

Procedure and Results: Confluent LR7 cell monolayers grown in 35-mmdishes were infected with each recombinant virus (8 PFU/cell) and viralinfectivity in culture media at different times post-infection (p.i.)was determined by titration on LR7 cells. TCID⁵⁰ (50% tissue cultureinfective doses) values were calculated and plotted (FIG. 4). Therecombinant viruses did not differ appreciably with respect to theinduction of extensive syncytia or cytopathic effects or in their plaquesize. However, MHV-□Δ45a and MHV-min differed from MHV-WT and MHV-□Δ2aHEin their one-step growth kinetics (FIG. 4). The two viruses displayedapproximately 10-fold lower titers at all time points.

Conclusion: All deletion viruses multiply well in vitro although thedeletion of genes 4 and 5a had a slightly negative effect.

I.1.e. Virulence of Recombinant Viruses in Mice:

Aim: Establishing whether the genetic deletions affect viral virulence.

Procedure and Results: The recombinant viruses were characterized intheir natural host, the mouse. As a first step, we determined thevirulence of the recombinant viruses. An LD⁵⁰ (50% lethal dose) assaywas carried out by inoculating MHV-negative, C57B1/6 mice miceintracranially with four 10-fold serial dilutions (5×10⁵⁻5×10²) ofrecombinant viruses. Viruses were diluted using PBS containing 0.75%bovine serum albumin. A volume of 25 μl was used for injection into theleft cerebral hemisphere. Five animals per dilution per virus wereanalyzed. LD⁵⁰ values were calculated by the Reed-Muench method based ondeath by 21 days post-infection. Clearly, deletion mutant viruses wereattenuated when compared to the recombinant MHV-WT. While the MHV-WTvirus had an LD⁵⁰ of 1.8×10⁴ no LD⁵⁰ could be derived for the deletionmutants. Although the animals inoculated with the higher doses showedsome signs of illness, none of the animals infected with any of thedeletion mutant viruses died up to input of 50,000 PFU/mouse. Thisimplies that the LD₅₀ for these viruses exceeds a value of 50,000 andmay well be above 100,000.

FIG. 25 illustrates the kinetics of mortality of mice infected with thehighest inoculation dose, 2.5×10⁵ plaque forming units (PFU), of WT anddeletion viruses. While all the animals inoculated with wild type virushad died by seven days post infection, the deletion viruses were highlyattenuated, displaying no death and less severe clinical symptoms.Despite the observation of no mortality, all mice infected with the □45aand □2aHE viruses showed clinical signs of hunched posture, disheveledappearance and waddling gait during the first weekpost-infection; thesesymptoms were less severe and observed in fewer mice infected withMHV-min.

Conclusion: Viruses with deletions of the sequences specifying the genes2a+HE or genes 4a+4b+5a or of the combination of all these genes exhibita significantly attenuated phenotype in mice. In other words, thenonessential genes of coronaviruses are not crucial for in vitro growthbut determine viral virulence. The attenuation acquired by theirdeletion thus provides excellent viral vaccines and therapeutic vectors.

I.2. Generation of Recombinant Viruses with Rearranged Gene Order:

General aim: establish whether the invariable order of the genesspecifying the polymerase functions (ORF1a/1b) and the structuralproteins S, E, M, and N in the coronaviral genome is essential for theviability of these viruses or whether rearrangement of this order istolerated.

I.2.a. Construction of Recombinant Viruses with Rearranged Gene Order:

Specific aim: generate MHV-A59 mutants in which the relative positionsof structural protein genes in the MHV-A59 genome are changed by movingthe M and/or E gene.

Approach: targeted RNA recombination using fMHV and synthetic donor RNAscarrying the intended rearrangements (FIG. 5, top).

Procedure: Transcription vectors for the production of donor RNA fortargeted recombination were constructed from plasmids pMH54 and pXH□2aHE(described above). In order to generate transcription vector pXHSM45N(FIG. 5, lower left part), a PCR product was generated by splicingoverlap extension (SOE)-PCR that contained the 3′ end of the M gene andthe 5′end of the N gene and in which a EcoRV restriction site wasintroduced between the M gene and IGS just upstream of the N gene. Togenerate this PCR fragment, outside primer 1C(5′-GTGTATAGATATGAAAGGTACCGTG-3′) (SEQ ID NO:12) corresponding to theregion of the M gene that contains the unique KpnI site, and outsideprimer 1097 (5′-CGAACCAGATCGGCTAGCAG-3′) (SEQ ID NO:13), correspondingto the region of the N gene that contains the unique NheI site, wereused. Primer 1095 (5′-AGATTAGATATCTTAGGTTCTCAACAATGCGG-3) (SEQ ID NO:14)and primer 1096 (5′-GAACCTAAGATATCTAATCTAAACTTTAAGGATG-3′) (SEQ IDNO:15) were used as inside primers. They correspond to the sequencebetween the M and the N gene and introduce the EcoRV restriction site.The resulting PCR product was cloned into pGEM-T easy (Promega) yieldingvector pXH0302. As a next step in the construction of pXHSM45N, pMH54was treated with the restriction enzymes Sse8387I and EcoRV and theresulting fragment was cloned into the EcoRV site of pXH0302 after beingblunted by T4 DNA polymerase treatment, yielding vector pXH0902. Afterexcision of the Sse8387I-EcoRV fragment of pMH54, the remaining vectorwas also blunted by T4 DNA polymerase treatment and religated resultingin plasmid pXH1401. Finally, plasmid pXH0902 was treated withrestriction enzymes NheI and BssHII and the resulting fragment wascloned into pXH1401 treated with the same enzymes, yielding pXHSM45N.

For the construction of pXHMSmN, first the ORFs 4, 5 and M were removedfrom pMH54 by restriction of this vector with enzymes Sse8387I andBssHII, followed by treatment with T4 DNA polymerase and religation ofthe remaining vector, which yielded pXH□45M5′. Next, the fragmentresulting from treatment of pXH0302 with enzymes NheI and BssHII wascloned into pMH54 treated with the same enzymes, resulting in pXHMeN.Subsequently, the fragment obtained after restriction of pXHMeN withEcoRV was cloned into pXH1802 (described above), which was digested withHindIII and treated with Klenow fragment of DNA polymerase I, yieldingpXH0305B. The fragment obtained by restriction of pMH54 with enzymesMluI and EcoRV was cloned into pB59 (2) treated with the same enzymes,which resulted in vector pXH2801. Next, the fragment resulting from thetreatment of pXH2801 with restriction enzymes KpnI and PstI was treatedwith T4 DNA polymerase and cloned into pXH1802 treated with restrictionenzyme HindIII and with Klenow fragment of DNA polymerase I, yieldingpXH0806. Subsequently, the fragment obtained by digestion of pXH0305Bwith SpeI and AflII was cloned into pXH0806 treated with the sameenzymes, resulting in pXH1506. Finally, pXHSmN was obtained by cloningthe fragment resulting from restriction of pXH1506 with RsrII and AvrIIinto pXH□45M5′ treated with the same enzymes.

For the construction of transcription vector pXH1bMS, vector pXHMeN wasrestricted with EcoRV. The resulting fragment was removed and the vectorwas religated yielding pXH□M. Next, the fragment obtained by digestionof pXH0305B with RsrII and AvrII was cloned into pXH□M treated with thesame enzymes, yielding pXH1bMS.

All constructs were confirmed by restriction and/or sequence analysis.They are depicted schematically in FIG. 5 (left). All new junctionsgenerated, including the introduction of the Sse8387I site downstream ofthe S gene in pMH54 (7), are indicated with arrowheads, while theirsequences are shown in Table 1. Recombinant viruses were generated byRNA-RNA recombination between transcription vector run-off transcriptsand the fMHV genome as described above. After 2 rounds of plaquepurification on LR7 cells the viruses were analyzed by reversetranscriptase-PCR on genomic RNA and found to contain the genomes withthe expected organization.

Conclusion: The strict gene order of the coronaviruses is not anessential prerequisite for viability.

I.2.b. RNA Synthesis by MHV Mutants with Rearranged Gene Order:

Aim: Confirming the patterns of RNAs synthesized in cells infected bythe mutant viruses.

Procedure: Infected 17Cl1 cells were metabolically labeled with[³³P}orthophosphate in the presence of actinomycin D essentially asdescribed (4, 10). Samples of total cytoplasmic RNA, purified usingUltraspec reagent (Biotecx), were denatured with formaldehyde andformamide, separated by electrophoresis through 1% agarose containingformaldehyde, and visualized by fluorography (FIG. 26A).

Result: Coronaviruses express their genome via the generation of a 3′co-terminal nested set of sg RNAs. Recombinant viruses with a rearrangedgenome organization are therefore predicted to synthesize patterns ofviral RNAs that are distinctly different from that of the parent virus.For the reconstructed wild-type virus (MHV-WT) and for MHV-□2aHE, theRNA patterns and the amounts of the genomic (g) and sg RNA speciesappeared to be similar to those observed previously (FIG. 3). Note thatMHV-□2aHE—as well as its derivatives MHV-MSmN and MHV-1bMS—does notsynthesize the sg RNA species encoding the 2a protein. For the MHVmutants with the rearranged genomes, all variant sg RNAs had mobilitiesthat corresponded to their predicted sizes (FIG. 26A and Table 4), andno obvious additional species were observed. MHV-SM45N grew very poorly,and was therefore labeled only weakly. All sg RNAs could be detectedexcept the one from which the M protein should be translated. The lowabundance of this sg RNA, the reason of which is unknown, is likely tobe the cause of the impaired growth of this virus. Overall, the patternsof viral RNAs synthesized by the cells infected with the recombinantviruses nicely reflect the changes made to the coronavirus genomeorganization.

Conclusion: The results confirm the genotypes of the recombinant virusesand demonstrate their expected phenotypes at the RNA level.

I.2.c. Tissue Culture Growth Phenotype:

Aim: Comparing the in vitro growth phenotypes of the mutant viruses.

Procedure and Results: Confluent LR7 cell monolayers grown in 35-mmdishes were infected with each recombinant virus (8 PFU/cell) and viralinfectivity in culture media at different times post-infection wasdetermined by titration on LR7 cells. TCID⁵⁰ values were calculated andplotted (FIG. 6). All viruses except the mutant MHV-SM45N were analyzedin this way. The infectious titer of this latter virus was too low(approximately 1000 times lower than the WT recombinant MHV) to performa one-step growth curve. Although MHV-1bMs appeared to induce syncytiasomewhat slower than the WT recombinant, all viruses replicatedapproximately to the same extent in the one-step growth curve.

Conclusion: Except for mutant virus MHV-SM45N, the gene rearrangementhad no dramatic effect on their in vitro growth characteristics.

I.2.d. Replication of Recombinant Viruses in Mice:

Aim: Establishing whether viruses with rearranged gene order are able toreplicate in mice.

Procedure and Results: Eight week old, MHV-negative, female BALB/c micewere used in the experiment. Viruses were diluted in PBS and a totalvolume 100 μl (10⁶ TCID⁵⁰) was used for injection in the peritonealcavity. Four animals per virus were inoculated. Mice were sacrificed andthe livers were removed at day 4 post-infection. The livers were placedin 1.5 ml DMEM, weighed and then frozen at −80° C. until tittered forvirus. Virus titers were determined by plaque-assay on LR7 cellmonolayers following homogenization of the organs. The replication ofMHV-WT, MHV-□2aHE and MHV-MSmN was studied in their natural host, themouse. While the 50% lethal dose of MHV-WT in mice was previouslydetermined at 2.7×10⁴ PFU, MHV-□2aHE was not virulent enough for a 50%lethal dose value determination (section I.1.e.). Therefore, we nowdecided to analyze the in vivo replication of the recombinant viruses.Mice inoculated intraperitoneally with 1×10⁶ TCID⁵⁰ were euthanized atday 4 post-infection and the viral replication in the liver wasdetermined. The results are shown in FIG. 26B. MHV-U□2aHE and MHV-MSmNreplicated in the liver to a similar extent albeit much lower thanMHV-WT. Deletion of ORFs 2a and HE generated a recombinant virus(MHV-□2aHE) that was attenuated in the natural host, as shown in sectionI.1.e., while additional rearrangement of the coronavirus gene order(MHV-MSmN) did not result in a more attenuated phenotype in this assay.

Conclusion: Viruses with a rearranged gene order, which lack the typicalcoronavirus genome organization, and viruses that lack ORFs 2a and HEare able to replicate in their natural host, the mouse.

I.3. Generation of Recombinant Viruses Expressing Foreign Genes:

Aim: Establish whether foreign genes can be inserted at differentpositions in the viral genome, either as an additional gene or replacingdeleted nonessential genes or in combination with a rearranged geneorder; establish whether these genes are expressed and whether they arestably maintained during in vitro passage of the virus.

I.3.a. Construction of Recombinant Viruses Carrying Reporter Genes:

Aim: Generate MHV-A59 viruses with foreign gene insertions.

Procedure: Several viruses were constructed containing a foreignreporter gene in their genome at different positions (see FIG. 7A). Tworeporter genes were used, encoding renilla luciferase (RL) and fireflyluciferase (FL). For both genes, a plasmid was constructed in which thegene was preceded by the MHV intergenic sequence (IGS). From thisconstruct the expression cassette (gene plus IGS) could then betransferred into the different transcription vectors.

As a first step, the MHV IGS was cloned in front of the RL gene. To thisend, primer 1286 (5′-GGATACTAATCTAAACTTTAG-3′) (SEQ ID NO:16) and 1287(5′-CTAGCTAAAGTTTAGATTAGATATCCTGCA-3′) (SEQ ID NO:17) were annealed toeach other and cloned into pRL-null (Promega) treated with NheI andPstI, resulting in pXH1909. For the construction of the transcriptionvector containing the RL gene between genes 2a and S (pXH22aRLS), thefollowing steps were taken. Vector p⁹⁶ (1) was restricted with HindIIIand MluI and the resulting fragment was cloned into pXH1802 (describedabove) treated with HindIII and BssHII, yielding pXH2103. Next, the RLexpression cassette was removed from pXH1909 by restriction with EcoRVand XbaI, treated with Klenow fragment of DNA polymerase I and clonedinto pXH2103 digested with HindIII and treated with Klenow fragment,resulting in pXH2509A. Finally, pXH22aRLS was constructed by cloning thefragment resulting from digestion of pXH2509A with RsrII and AvrII intopMH54 treated with the same enzymes.

For the construction of pXH2ERLM the same expression cassette that wasused to make pXH2509A, was cloned into pMH54 digested with EcoRV.

This same expression cassette was also cloned into pXHMeN (describedabove) treated with EcoRV, yielding pXH2ERLN. Subsequently, pXH2MRLN wasconstructed by cloning the fragment resulting from restriction of pXHMeNwith EcoRV into pXH2ERLN treated with the same enzyme. pXHMSmNRL wasconstructed by cloning the renilla expression cassette into pXHMSmN(described above) treated with EcoRV.

For the construction of pXHEFLM, the FL gene was first cloned behind thesame IGS as was used for the RL expression cassette. To this end, theluciferase gene was removed from pSP-Luc+ (Promega) by restriction withAvrII and XbaI and cloned into pXH1909 treated with NheI and XbaI,yielding pXH2711. Subsequently, the FL expression cassette was cut outof pXH2711 by restriction with EcoRV and XbaI, treated with Klenowfragment of DNA polymerase I, and cloned into pMH54 restricted by EcoRV,resulting in pXHEFLM.

Plasmid pXHminFL was constructed by cloning the FL expression cassetteinto pXHmin (described above) digested with EcoRV. This plasmid lacksORFs 2a/HE/4a/4b/5a and contains the FL gene between genes E and M.

After confirmation of all constructs by restriction and sequenceanalysis, recombinant viruses were generated by RNA-RNA recombinationbetween transcription vector run-off transcripts and the fMHV genome asdescribed above. The resulting viruses were genetically confirmed byRT-PCR analysis.

Results of recombinant viruses containing the RL gene are shown in FIGS.7B and 27. To confirm the insertion of this gene an RT step wasperformed on genomic RNA with primer 1412 (5′-CTGCGGACCAGTTATCATC-3′)(SEQ ID NO:18) which is complementary to the 5′ end of the RL gene.Subsequently, a PCR was performed with primer 1091(5′-GTTACAAACCTGAATCTCATCTTAATTCTGGTCG-3′) (SEQ ID NO:19) and primer1413 (5′-CATCCGTTTCCTTTGTTCTGG-3) (SEQ ID NO:20) for MHV-ERLM andMHV-MRLN or with primer 1173 (5′-GACTTAGTCCTCTCCTTGATTG-3) (SEQ IDNO:21) and primer 1413 for MHV-2aRLS.

Primer 1091 and primer 1173 correspond with the 3′ end of gene 4b andgene 1b, respectively, while primer 1413 corresponds with the 5′ end ofthe RL gene. As positive controls, the appropriate transcription vectorswere taken along. In all cases, PCR fragments were obtained of the samesize as the positive controls, while the water control was negative. Theobserved (and predicted) fragment sizes were approx. 1,000 bp forMHV-2aRLS, approx. 670 bp for MBV-ERLM, and approx. 1,300 bp forMHV-MRLN (7B). For MHV-MSmNRL a PCR was performed with primer 1091 andprimer 1413 and with primer 1173 and primer 1413. As positive controls,the appropriate transcription vector was taken along. In all cases, PCRfragments were obtained of the same size as the positive controls. Theobserved (and predicted) fragment sizes were approx. 670 bp for the PCRreaction with primers 1091 and 1413, and approx. 1,200 bp for the PCRreaction with primers 1173 and 1413 (7B) (note that in FIGS. 7B and 27for each type of virus 2 independently obtained, viral clones wereanalyzed and included). The results confirmed the insertion of the RLgene into the MHV genome at the correct position.

Results of recombinant viruses containing the FL gene are shown in FIG.28. To confirm the insertion of this gene an RT step was performed ongenomic RNA with primer 1475 (5′-GCCTAATGCAGTTGCTCTCC-3) (SEQ ID NO:22)which is complementary to the 5′ end of the FL gene. Subsequently, a PCRwas performed with primer 935 (5′-GTTTTAGCACAGGGTGTGGCTCATG-3) (SEQ IDNO:23), which corresponds with the 3′ end of the S gene, and primer 1474(5′-CCATCTTCCAGCGGATAG-3) (SEQ ID NO:24), which corresponds with the 5′end of the FL gene. As positive controls, the appropriate transcriptionvectors were taken along. In all cases, PCR fragments were obtained ofthe same size as the positive controls, while the water control wasnegative. The observed (and predicted) fragment sizes were approx. 1,200bp for MHV-EFLM, and approx. 500 bp for MHV-minFL (note that in FIG. 28for each type of virus 2 independently obtained, viral clones wereanalyzed and included).

Conclusion: Insertion of genetic modules into the coronaviral genome istolerated at all positions tested; all the intended viruses were viable.

I.3.b. RNA Synthesis by Recombinant Viruses:

Aim: Confirming the patterns of RNAs synthesized in cells infected bythe mutant viruses.

Procedure: Infected 17Cl1 cells were metabolically labeled with[³³P]orthophosphate in the presence of actinomycin D essentially asdescribed (4, 10). Samples of total cytoplasmic RNA, purified usingUltraspec reagent (Biotecx), were denatured with formaldehyde andformamide, separated by electrophoresis through 1% agarose containingformaldehyde, and visualized by fluorography (FIGS. 29-31; note that thesubgenomic RNA species in this figure are designated by theircomposition, rather than as RNA2 through RNA7, since the numericaldesignations would be ambiguous for the mutants).

Result: For all the MHV mutants with the luciferase genes, all variantsg RNAs had mobilities that corresponded to their predicted sizes (FIGS.29-31). For the viruses containing the renilla luciferase gene noobvious additional species were observed.

For the viruses containing the firefly luciferase gene two additionalRNA species were observed, which correspond in size with transcriptionof sgRNAs from sequences in the firefly luciferase gene that are similarto the MHV IGS. Overall, the patterns of viral RNAs synthesized by thecells infected with the recombinant viruses nicely reflect the insertionof the luciferase expression cassettes into the coronavirus genome.

Conclusion: The results confirm the genotypes of the recombinant virusesand demonstrate their expected phenotypes at the RNA level.

I.3.c. Replication of Viruses Expressing Renilla or Firefly Luciferase:

Aim: Compare the growth characteristics of the viruses with wild-typevirus and with each other.

Procedure and Results: After two rounds of plaque purification virusstocks were prepared, titrated and used for high m.o.i (m.o.i. of 8)infection of LR7 cells after which the viral infectivities in theculture media were monitored. The results are represented by the growthcurves shown in FIGS. 8A and 8B, and by the results shown in FIG. 32. OfMHV-EFLM two viral clones, independently obtained from the recombinationexperiment described under I.3.a, were analyzed. In FIG. 32, the TCID⁵⁰values obtained at 8-9 hours post-infection are shown. Obviously, thegrowth characteristics of the recombinant viruses shown in FIGS. 8A and8B are essentially indistinguishable; all viruses grew to titers thatwere comparable to that of recombinant wild-type virus. MHV-MSmNRLreached somewhat lower titers (FIG. 32).

Conclusion: The inserted expression cassettes hardly affected the invitro growth characteristics of the recombinant viruses.

I.3.d. Expression of Renilla and Firefly Luciferase in Cell Culture:

Aim: Establish whether the inserted expression cassettes werefunctional.

Procedure and Results: Confluent monolayer cultures of LR7 cells wereinfected at an m.o.i. of 8 and the production of luciferase activity inthe cells was monitored over time. RL expression in cells was measuredby using the Dual-Luciferase Reporter Assay System (Promega) accordingto the manufacturer's instructions. Similarly, FL expression wasmeasured by using the Luciferase Assay System (Promega) according to themanufacturer's instructions. RL and FL activity was measured in relativelight units (RLU) using a luminometer (Lumac Biocounter M2500 or TurnerDesigns Model TD-20/20).

The results are shown graphically in FIGS. 9A, 9B and 33. Allrecombinant viruses except for the recombinant wild-type virus expressedhigh levels of luciferase, indicating that both the renilla and thefirefly luciferase gene cassettes were functional at each genomicposition tested. For most viruses, the highest expression level wasreached at 9 hours post-infection. The expression level of fireflyluciferase at this time-point was determined at 1.6 ug/10⁶ cells.MHV-minFL expressed levels of luciferase activity that were comparableto those produced by the other recombinant viruses containing the FLgene, while expression of the renilla luciferase gene in cells infectedwith MHV-MSmNRL was approx. 10-fold lower than in cells infected withMHV-ERLM. This difference corresponds with the difference found inreplication between MHV-MSmNRL and MHV-ERLM.

Conclusion: Foreign genes can be expressed by coronaviruses both by theadditional insertion of such a gene, by using the genetic space createdby deletion of nonessential genes, or in combination with a rearrangedgenome organization.

I.3.e. Maintenance of Foreign Genes During Viral Passage:

Aim: Evaluate the stability of the inserted luciferase genes duringviral passage.

Procedure and Results: The recombinant viruses MHV-ERLM and MHV-EFLMwere passaged 8 times over LR7 cells at low m.o.i. (<0.05). After eachpassage, the viral infectivity (TCID⁵⁰) in the culture medium at 9 hourspost-infection was determined. Subsequently, the firefly and renillaluciferase activity was determined in a parallel expression experiment(m.o.i.=5) at 8 hours post-infection (FIG. 10). Both of MHV-ERLM and ofMHV-EFLM two independently obtained clones A and B were analyzed. Whilethe renilla luciferase expression was consistently stable for at least 8passages, that of the firefly luciferase was stable only for 5 passages.After passage 5 of MHV-EFLM, the expression level clearly decreased forboth independent clones. After 8 passages, 10 viral clones were isolatedby plaque assay both of MHV-ERLM and of MHV-EFLM and tested forluciferase expression. While for MHV-ERLM all 10 clones were positive, 9of the MHV-EFLM clones no longer showed clearly detectable luciferaseexpression.

Conclusion: A foreign gene can be stably maintained in the coronaviralgenome for at least 8 passages in vitro.

I.3.f. Expression of Firefly Luciferase in Mice:

Aim: Establish whether the inserted luciferase gene is expressed in thenatural host, the mouse.

Procedure and Results: Eight weeks old, MHV-negative, female BALB/c micewere used in the experiment. Mice were inoculated intranasally with 10⁶TCID⁵⁰ MHV-EFLM. Four animals per virus were used. Mice were sacrificedand the livers and brains were removed at day 4 post-infection. Organswere quick-frozen in liquid nitrogen and homogenized in Cell CultureLysis Reagent provided with the Luciferase Assay System (Promega). FLactivity was measured according to the manufacturer's instructions usinga luminometer (Lumac Biocounter M2500). Clearly, as shown in FIG. 34,luciferase activity could be detected both in liver and brain. As analternative way to evaluate whether the foreign gene was also expressedin vivo, i.e., in animals, a mouse was inoculated intraperitoneally with10⁶ TCID⁵⁰ MHV-EFLM. Four days later the mouse was sedated andluciferain was administered subcutaneously. The luciferase expressionwas evaluated 5 minutes later by real time recording of the emission oflight from the body of the sedated mouse using a sensitive screencoupled to a CCD camera. Light emanating from the liver area of themouse was clearly observed.

Conclusion: A foreign gene can be expressed by coronaviruses in theirnatural host.

I.3.g. Generation of MHV Expressing Two Foreign Genes from One Genome:

Aim: Establish whether two foreign genes can be expressed from a singlegenome.

Procedure and Results: pXH2aRLSEFLM was generated by cloning the FLexpression cassette into pXH2aRLS digested with EcoRV. Afterconfirmation of the construct by restriction and sequence analysis,recombinant viruses were generated by RNA-RNA recombination betweentranscription vector run-off transcripts and the fMHV genome asdescribed above. The resulting virus, MHV-RLFL, was geneticallyconfirmed by RT-PCR analysis. Both renilla luciferase activity andfirefly luciferase activity could be detected in individual plaques.After generation of a high titer stock, confluent monolayer cultures ofLR7 cells were infected at an m.o.i. of 8 and the production ofluciferase activity in the cells was monitored over time. RL expressionin cells was measured by using the Renilla Assay System (Promega)according to the manufacturer's instructions. Similarly, FL expressionwas measured by using the Luciferase Assay System (Promega) according tothe manufacturer's instructions at 8 hours post-infection. RL and FLactivity was measured in relative light units (RLU) using a luminometer(Lumac Biocounter M2500). The results show that MHV-RLFL replicated tothe same extent as MHV-2aRLS and MHVEFLM (FIG. 46A). MHV-RLFL expressedboth renilla luciferase and firefly luciferase, MHV-2aRLS expressedrenilla luciferase only, while MHV-EFLM expressed firefly luciferaseonly (FIG. 46B).

Conclusion: Two foreign genes can be expressed from a single coronavirusgenome:

I.3.h. Generation of MHV Expressing a Chimeric Spike-GFP Gene:

Aim: Establish whether recombinant MHV can be generated that expressesand incorporates chimeric spike-GFP proteins.

Procedure and Results: The GFP gene was cloned in frame with the S genein pMH54. After confirmation of the construct by restriction andsequence analysis, recombinant viruses were generated by RNA-RNArecombination between transcription vector run-off transcripts and thefMHV genome as described above. The resulting virus, MHV-SGFP, wasgenetically confirmed by RT-PCR analysis. Plaques were microscopicallyanalyzed and showed expressing of GFP as evidenced by the greenfluorescence. Immunoprecipitation analysis indicated that hybridproteins were generated that could be precipitated with specificantibodies to MHV-S and GFP.

Conclusion: MHV can be generated that expresses a chimeric S-GFP gene instead of a wild-type S gene. This mutant virus is viable and could bepropagated in cell culture indicating that these hybrid proteins areincorporated into the viral particle.

I.4. Inhibition of Infection and of Cell Fusion by Spike Protein DerivedPeptide:

General aim: inhibit coronaviral infection and the spread of an ongoinginfection by interfering with membrane fusion using peptides.

Specific aim: produce a peptide constituting a sequence derived from themembrane-proximal heptad repeat region (HR2) of the MHV-A59 S proteinand demonstrate its inhibitory effect on MHV-A59 entry into LR7 cellsand on cell-cell fusion in an infected culture of these cells.

Procedures: a. Plasmid Constructions:

A PCR fragment from a template plasmid pTUMS (13) containing the MHV-A59spike gene was obtained, corresponding to amino acid residues 1216-1254(HR2) of the S protein (FIG. 20). The forward primer used was:5′-GCGGATCCATCGAAGGTCGTGATTTATCTCTCGATTTC-3′ (SEQ ID NO:25). This primerintroduced an upstream BamHI site and a sequence encoding a factor Xacleavage site immediately downstream of the BamHI site into theamplified fragment. The reverse primer(5′-CGAATTCATTCCTTGAGGTTGATGTAG-3′) (SEQ ID NO:26) contained adownstream EcoRI site as well as a stop codon preceding the EcoRI site.The PCR fragment was cloned into the BamHI-EcoRI site of the pGEX-2Tbacterial expression vector.

b. Bacterial Protein Expression and Purification:

Freshly transformed BL21 cells (NOVAGEN) were grown in 2YT medium to logphase (OD₆₀₀ of 1.0) and subsequently expression was induced by addingIPTG (GibcoBRL) to a final concentration of 0.4 mM. Two hours after thestart of induction the cells were pelleted, resuspended in 1/25 ofculture volume 10 mM Tris pH (8.0), 10 mM EDTA, 1 mM PMSF and sonicatedon ice (5 times for 2 min with 1-min intervals). Cell lysates werecentrifuged at 20,000×g for 60 minutes at 4° C. Then, 2 mlglutathione-sepharose 4B (50% v/v in PBS) was added per 50 ml ofsupernatant and the suspension was incubated overnight (O/N) at 4° C.under rotation. Beads were washed three times with 50 ml PBS andresuspended in a final volume of 1 ml PBS. Peptides were cleaved fromthe GST moiety on the beads using 20 U of thrombin by incubation for 4hours at room temperature (RT). Peptides in the supernatant were HPLCpurified on a Phenyl column with a linear gradient of acetonitrilecontaining 0.1% trifluoroacetic acid. Peptide containing fractions werevacuum-dried O/N and dissolved in water. Peptide concentration wasdetermined by measuring the absorbance at A₂₈₀ nm or by BCA proteinanalysis (Micro BCA™ Assay Kit, PIERCE).

c. Virus-Cell Entry and Cell-Cell Fusion Inhibition Assays:

The potency of the HR2 peptide in inhibiting viral infection wasdetermined using the recombinant MHV-EFLM expressing the fireflyluciferase. Confluent monolayers of LR7 cells in 96-well plates wereinoculated at 37° C. in DMEM at a multiplicity of infection of 5 for 1hour in the presence of varying concentrations of peptide ranging from0.4-50 μM. After 1 hour, cells were washed with DMEM and medium wasreplaced by peptide-free DMEM. At 5 hours post-infection cells werelysed for 15 minutes at RT in 50 μl Lysis buffer, according to themanufacturer's protocol (Luciferase Assay System, Promega). Upon mixingof 10 μl cell lysate with 40 μl substrate, luciferase activity wasmeasured immediately using a Wallace Betaluminometer. The 50% effectiveinhibitory concentration (EC₅₀ value) was calculated by fitting theinhibition data to an equilibrium binding equation: % luciferaseactivity=100/(1+(C/EC₅₀). The ability of the peptide HR2 to inhibitspike mediated cell-cell fusion was determined using a plaque assay.Monolayers of LR7 cells in 6-well plates were inoculated with 50 PFU ofMHV-A59 in DMEM at 37° C. After one hour, the cells were washed withDMEM and an agar overlay was added containing the HR2 peptide at 50, 10,2, 0.4 and 0.08 μM concentrations. Plaques were counted at 48 hourspost-infection, after staining and fixing with 0.9% formaldehyde/0.75%crystal violet.

Results: The potency of the HR2 peptide to inhibit virus entry wastested using a virus expressing a luciferase reporter gene as thisallows extremely sensitive detection of infection. Inoculations of cellswith the virus were carried out in the presence of differentconcentrations of HR2 peptide. After 1 hour of inoculation, cells werewashed and incubated further in culture medium in the absence ofpeptide. At 4 hours post-infection, before syncytium formation normallytakes place, cells were lysed and tested for luciferase activity (FIG.11). In this figure the normalized luciferase activity, representing thesuccess of infection, was plotted against the peptide concentrationpresent during inoculation. The HR2 peptide blocked viral entry veryefficiently, infection being inhibited virtually completely at theconcentration of 50 μM. The effective concentration (EC₅₀) at which 50%of viral infection was inhibited was 0.15 μM. The ability of the HR2peptide to inhibit cell-cell fusion mediated by the spike protein wasexamined by using a plaque assay. After inoculation of (parallelcultures of) cells in the absence of peptide, an overlay was appliedcontaining different concentrations of HR2 peptide. The formation ofplaques appeared to be completely abolished in the presence of HR2peptide concentrations of up to 0.4 μM (FIG. 12). At 0.08 μM of the HR2peptide only tiny plaques could be observed.

The specificity of the inhibition was demonstrated in all these assaysby testing in parallel the effect of other peptides (FIG. 20) preparedidentically and used at the same concentrations, including, forinstance, the peptide corresponding to amino acids 1003-1048 of theMHV-A59 S protein (shown as “control peptide” in FIG. 11). Only the HR2peptide was effective.

Conclusion: The HR2 peptide is a potent inhibitor both of virus entryinto cells and of MHV-A59 spike mediated cell-cell fusion.

II. Feline Infectious Peritonitis Virus (FIPV), Strain 79-1146:

II.1. Generation of mFIPV, a Feline Coronavirus Growing on Murine Cells:

General aim: To set up a targeted RNA recombination system for felinecoronavirus FIPV 79-1146 similar to the one described above for thegenetic manipulation of the murine coronavirus MHV-A59.

Specific aim: Generate mFIPV, an FIPV derivative in which the spikeprotein ectodomain has been replaced genetically by that of the MHV-A59S protein, thereby shifting the tropism of the chimeric virus to murineinstead of feline cells.

I.1.a. Construction of a Synthetic RNA Transcription Vector:

Aim: Prepare a plasmid construct from which synthetic donor RNA can betranscribed for targeted RNA recombination and which consists ofsequences derived from the 5′ end of the FIPV genome fused to sequencesderived from the 3′ part of the viral genome, i.e., all sequencesdownstream of (and including) the 3′-terminal end of the ORF1B. Also:prepare a derivative of this plasmid in which the sequence encoding thespike ectodomain has been replaced by that encoding the correspondingdomain of the MHV-A59 spike protein.

Procedure and Results: Using standard DNA cloning techniques the vectorpBRDI1 was constructed which contains a cDNA copy of the 5′-most 702bases ligated to the 3′-most 9.262 bases of the FIPV 79-1146 genome(FIG. 13A). The ligation was done in such a way that the ORF1A (pol 1A)gene fragment was fused in frame at its 3′-end to the 5′-end of the ORF1B (pol 1B) gene fragment (FIG. 14B). Furthermore, at this point aunique SacI restriction site was introduced (FIG. 14B). The felinecoronavirus sequence was placed under the control of a bacteriophage T7polymerase promoter sequence followed by a triple G (FIG. 14A) to driveefficient in vitro RNA transcription using the T7 polymerase. At the3′-end, the sequence was terminated by a polyA tail of 15 A's followedby a unique NotI restriction site (FIG. 14C) to facilitate run-offtranscription. The T7 promoter sequence is preceded by a unique XhoIrestriction site (FIG. 14A) such that the feline coronavirus cDNA couldbe cloned as a XhoI-NotI restriction fragment of 10.015 bp into thebackbone vector pBRXN (11) resulting in pBRDI1 (FIG. 13A).

Plasmid pBRDI1 was subsequently used to prepare pBRDI2 in which the FIPVS gene was replaced by a chimeric spike gene (mS) composed of a partencoding the ectodomain of the MHV spike protein and a part encoding thetransmembrane and endodomain of the FIPV spike protein. To introducethis hybrid gene into pBRDI1, first the 3′ end of the feline pol1B genewas fused to the 5′ end of the murine spike gene (see FIG. 14D forsequence at junctions). This fusion product was cloned into pTMFS (12)as a SacI-StuI fragment, resulting in pTMFS1 (FIG. 13B). The hybrid genewas then isolated from pTMFS1 as a SacI-AlfII fragment and used toreplace the FIPV spike gene from pBRDI1 resulting in pBRDI2 (FIG. 13B).The sequences of pBRDI1 and pBRDI2 are shown in addendum 1 and 2,respectively.

II.1.b. Generation of mFIPV by RNA Recombination:

Aim: Generate mFIPV, an FIPV derivative targeted to murine cells.

Procedure and Results: Capped, run-off donor RNA transcripts weresynthesized from NotI-linearized pBRDI2 using a T7 RNA polymerase kit(Ambion) according to the instructions of the manufacturer. Thetranscripts were introduced into feline FCWF cells (80 cm² cultureflask) that had been infected before with FIPV 79-1146 (m.o.i. of 1), byelectroporation (Gene pulser electroporation apparatus, Biorad, 2consecutive pulses; 0.3 kV/960 microF). The electroporated cells werecocultured in a 25 cm² flask with murine LR7 cells (50% confluency) toallow recombination of the synthetic and genomic RNA (FIG. 15). After 24hours of incubation at 37° C., massive syncytia could be observed ofboth murine LR7 cells and feline FCWF cells. Candidate mFIPVrecombinants were selected by taking off the culture supernatant andpassaging the virus by three consecutive end-point dilutions on LR7cells. The resulting virus was unable to infect and cause cytopathiceffect on FCWF cells.

Conclusion: A virus with the intended murine cell tropism was obtained.

II.1.c. mFIPV Protein Analysis:

Aim: Confirm the identity of mFIPV at the level of viral proteinsynthesis.

Procedure and Results: Murine LR7 cells were infected with mFIPV and theproteins were labeled with ³⁵S-labeled amino acids for 2 hours startingat 5 hours post-infection As controls, we infected LR7 and FCWF cellswith MHV and FIPV, respectively, and labeled them similarly from 5-7hours post-infection. After the labeling, cell lysates were prepared andimmunoprecipitations were carried out in the presence of detergent asdescribed (12). The following antibodies were used (for references, see12): G73 (αFIPV), an ascitis fluid obtained from an FIPV-infected cat(provided by H. Vennema); K134 (αMHV), a rabbit serum raised againstpurified MHV-A59; WA3.10 (αS_(m)), a Mab against an epitope present inthe MHV-A59 S ectodomain; 23F4.5 (αS_(f)), a Mab against an epitope inthe FIPV S ectodomain. The immunoprecipitated proteins were taken up inelectrophoresis sample buffer and heated for 2 minutes at 95° C. exceptfor one protein sample (lane 4 in FIG. 16) which was kept at roomtemperature to prevent aggregation of the MHV M protein. The proteinswere analyzed by electrophoresis in SDS-12.5% polyacrylamide gel. Theelectrophoretic patterns are shown in FIG. 16. As expected, theanti-FIPV antibodies precipitated the FIPV proteins S, M and N from thelysate of FIPV-infected cells (lane 10), but none of the MHV proteinsfrom lysate of MHV-infected cells (lane 1). The 23F4.5 Mab precipitatedthe feline S of FIPV (lane 11) but not the murine S of MHV (lane 2), asexpected. Also, the anti-MHV serum precipitated the MHV proteins S, Nand M (lane 3 and 4) but not the FIPV proteins (lane 12), and the WA3.10Mab precipitated the MHV S protein (lane 5) but not the FIPV S protein(lane 13). When looking at the proteins precipitated from themFIPV-infected cell lysates, it is clear that the anti-FIPV serum G73precipitated the M and N proteins, but not the S protein (lane 6). Sprotein was also not precipitated by the 23F4.5 Mab (lane 7). The mFIPVS protein was, however, precipitated by the anti-MHV serum (lane 8) aswell as by the Mab WA3.10 (lane 9).

Conclusion: Cells infected by mFIPV express the predicted viralproteins, particularly the hybrid S protein with the MHV-derivedectodomain.

II.1.d. mFIPV Growth Characteristics:

Aim: Compare titers obtained for mFIPV with those for FIPV and MHV-A59.

Procedure and Results: Infection and titration experiments revealed thatthe recombinant virus mFIPV was no longer able to infect feline FCWFcells but did grow efficiently in murine LR7 cells showing similargrowth characteristics as MHV-A59. This is demonstrated, for instance,by a comparison of their one-step growth curves in these cells shown inFIG. 17. In this experiment, cells were infected with mFIPV and MHV-A59(m.o.i. of 5 each) after which samples from the culture fluid were takenat various time points and titrated on LR7 cells by end-point dilution.Also, mFIPV induced extensive syncytia and cytopathic effects uponinfection of these cells. Stocks of the recombinant mFIPV grown in LR7cells reached titers of the same order of magnitude as FIPV did in FCWFcells (5.10⁷ PFU/ml). This was, however, an order of magnitude lowerthan was observed for MHV-A59 in LR7 cells.

Conclusion: The replacement of the spike protein ectodomain has resultedin a recombinant mFIPV that replicates well in its “new” host cells andhas apparently not lost much of its biological fitness.

II.2. Generation of Live Attenuated FIPV Vaccine by Gene Deletions:

General aim: Delete gene sequences from the FIPV genome that arenonessential for viral replication in vitro to obtain deletion virusesthat are attenuated in feline animals and are therefore viral (vector)vaccine candidates.

II.2.a. Construction of Synthetic RNA Transcription Vectors:

Aim: Construct the plasmids for the synthesis of donor RNA transcriptslacking the genes 3ABC and/or 7AB for targeted recombination with mFIPVRNA (FIG. 18, top part).

Procedure and Results: Deletions of genes 3ABC and 7AB were introducedinto the plasmid pBRDI1 using SOE-PCR. For the primers used, see Table 2and FIG. 18, left side.

To delete the 3ABC cluster, combinations of primers 1 and 4 and of 2 and3 were used to generate fragments of 375 bp (A) and 1012 bp (B),respectively (FIG. 18, left side). Fragments A and B were fused usingthe overlap between both fragments through primers 3 and 4, andamplified using primers 1 and 2 resulting in a 1366 bp fragment (C).Fragment C was digested with AflII and SnaBI and cloned into AflII andSnaBI-digested pBRDI1, resulting in pBRDI1Δ3ABC (FIG. 18).

To delete the 7AB genes, combinations of primers 5 and 8 and of primers6 and 7 were used to generate fragments of 1215 bp (D) and of 324 bp(E), respectively (FIG. 18). Fragments D and E were fused using theoverlap between both fragments through primers 7 and 8, and amplifiedusing primers 5 and 6 resulting in a 1524 bp fragment (F). Fragment Fwas digested with MluI and NotI and cloned into MluI and NotI-digestedpBRDI1, resulting in pBRDI1Δ7AB (FIG. 18). The correctness of thesequences of fragments C and F was confirmed by DNA sequencing.

To construct pBRDI1Δ3ABC+Δ7AB, the 1524 bp MluI/NotI fragment ofpBRDI1Δ7AB was introduced into MluI/NotI-digested pBRDI1Δ3ABC (FIG. 18).

Conclusion: The pBRDI1-derived donor RNA constructs lacking the geneclusters 3ABC and/or 7AB were obtained.

II.2.b. Generations of Recombinant FIPVs Lacking Genes:

Aim: Generate the recombinant FIPVs that lack the sequences for the 3ABCgenes, for the 7AB genes, and for both these gene clusters.

Procedure and Results: Capped, run-off donor transcripts weresynthesized from NotI-linearized pBRDI1, pBRDI1Δ3ABC, pBRDI1Δ7AB andpBRDI1Δ3ABC+Δ7AB, respectively, using a T7 RNA polymerase kit (Ambion)as specified by the manufacturer. The donor transcripts were introducedinto murine LR7 cells (80 cm² flask), that had been infected before withmFIPV (m.o.i. of 0.4), by electroporation (Gene pulser electroporationapparatus, Biorad, 2 consecutive pulses; 0.85 kV/50 microF). Theelectroporated cells were cocultured in a 25 cm² culture flask withfeline FCWF cells (50% confluency). After 24 hours incubation at 37° C.massive syncytia could be detected in both the murine LR7 cells and thefeline FCWF cells. Candidate deletion viruses released into the mixedcell culture supernatant were purified by two rounds of plaquepurification on FCWF cells.

Conclusion: Viruses that had acquired the ability to grow in feline FCWFcells had been obtained from the recombination experiment with mFIPV.

II.2.c. Genetic Analysis of Recombinant FIPVs Lacking Genes:

Aim: Confirm the genetic make-up of the putative deletion viruses.

Procedure and Results: To evaluate whether the intended deletions indeedoccurred in the various FIPV deletion mutants, RT-PCR on the genomicviral RNA's was performed focusing on the 3ABC and the 7AB region. Theprimers used and DNA sizes expected are indicated in Table 2 and FIG. 18(right side). The results of the RT-PCR analyses are shown in FIG. 19.FIG. 19A reveals that the recombinant wild-type virus (r-wtFIPV) andFIPVΔ7AB are each carrying the 3ABC region whereas this region islacking in the viruses FIPVΔ3ABC and FIPVΔ3ABC+Δ7AB, as judged by thesizes of the amplified fragments. FIG. 19B demonstrates that r-wtFIPVand FIPVΔ3ABC are still carrying the 7AB region whereas the virusesFIPVΔ7AB and FIPVΔ3ABC+Δ7AB are lacking this region. The 397 bp and 646bp fragments, indicative of a 3ABC and 7AB deletion, respectively, werecloned and sequenced which confirmed the expected DNA sequences.

Conclusion: The deletion viruses had precisely the intended genomicdeletions.

II.2.d. Growth Characteristics of FIPVs Lacking Genes

Aim: Check cell tropism of the deletion viruses and evaluate their invitro growth.

Results: All 4 recombinant viruses were inoculated onto mouse LR7 cellsbut failed to produce any cytopathic effects. They did, however, growefficiently in feline FCWF cells, as expected. The viruses r-wtFIPV,FIPVΔ3ABC and FIPVΔ7AB reached titers that were similar to thoseobtained with the parent FIPV strain 79-1146 on these cells. The titerswere all in the order of 5.10⁷ PFU/ml (FIG. 40). However, the doublemutant FIPVΔ3ABC+Δ7AB grew less efficient; titers obtained with thisvirus were generally 1 to 2 log units lower (FIG. 40).

Conclusion: The sequences comprising the gene clusters 3ABC and 7AB arenot essential for the viability of FIPV. Apparently, neither thenucleotide sequences nor the proteins encoded by the genes are essentialfor the replication of the virus.

II.2.e. Virulence of Recombinant Viruses in Cats:

Aim: Establishing whether the genetic deletions affect virulence.

Procedure and Results: The recombinant deletion viruses werecharacterized in their natural host, the cat. To this purpose, 24 SPFcats (5 months old) were placed into 5 groups and inoculated oronasally(100 pfu) with FIPV strain 79-1146 (n=4), r-wtFIPV (n=5), FIPVΔ3ABC(n=5), FIPVΔ7AB (n=5) and FIPVΔ3ABC+Δ7AB (n=), respectively, andfollowed for (at least) 3 months. Clinical disease signs were scored asshown in Table 5. Inoculation of the cats with the deletion variants didnot induce clinical signs of disease. Rather, all cats remained totallyhealthy throughout the experiment (Table 6 and FIG. 35). In contrast,infection with the wild type controls FIPV 79-1146 and r-wtFIPV induceda rapid onset of clinical disease characterized by depression, anorexia,jaundice, weight loss and leukopenia (Table 6). Three out of four andfive out of five cats inoculated with FIPV 79-1146 and r-wtFIPV,respectively, had to be euthanized due to advanced symptoms of FIPbetween day 14 and day 42 after infection (FIG. 35).

Conclusions: 1) FIPV 79-1146 and its wild type recombinant equivalentr-wtFIPV are equally virulent; and 2) Viruses with deletions of thesequences specifying the genes 3abc or 7ab or of the combination of someor all these genes exhibit a significantly attenuated phenotype in cats.

II.2.f. Immune Response Induced by Recombinant Viruses:

Aim: Determining the FIPV-neutralizing activity in cat sera.

Procedure and Results: FIPV-specific antibody responses of the catsinoculated with the deletion viruses were characterized. To thispurpose, blood samples were obtained at days 0, 21 and 90post-infection, and heat-inactivated sera were prepared and incubatedwith FIPV 79-1146 (10.000 PFU) after which the FIPV-neutralizingactivity was determined using FCWF cells in a 96-well microplate assay.Titers were expressed as the reciprocal of the lowest dilution that nolonger inhibited viral cytopathic effects. As expected, at day 0 none ofthe cat sera showed a significant FIPV-neutralizing activity. At day 21,all cats had sero-converted and showed high titers of neutralizingantibodies. The titers observed in cats inoculated with FIPV 79-1146,r-wtFIPV, FIPVΔ3ABC and FIPVΔ7AB were comparable whereas the titersobserved in FIPVΔ3ABC+Δ7AB infected cats were approximately 50 foldlower (FIG. 36). Overall, the titers remained high for at least 90 days(end of experiment).

Conclusion: Despite the absence of clinical disease signs, high titersof neutralizing antibodies are observed in cats that had been inoculatedwith FIPV deletion variants. This strongly suggests that the deletionviruses are viable and replicate in the cat leading to a strong immuneresponse in the form of an antibody response.

II.2.g. FIPV Deletion Viruses Serve as Attenuated, Live Vaccines:

Aim: To study whether previous inoculation with the attenuated deletionvariants protects the cats against a FIPV 79-1146 challenge.

Procedure and Results: The cats previously inoculated with theattenuated deletion variants were challenged oranasally with FIPV79-1146 (100 pfu) at day 90. As a control group, 4 untreated cats ofsimilar age were challenged identically. The control group showed arapid onset of clinical disease characterized by depression, anorexia,jaundice, weight loss and leukopenia (day 7). A similar rapid onset ofsymptoms was observed with 3 out 5 cats previously inoculated withFIPVΔ3ABC+Δ7AB, whereas all cats previously infected with FIPVΔ3ABC orFIPVΔ7AB remained generally healthy and without typical FIP symptomsthroughout the experiment (Table 7), although 2 out 5 cats previouslyinoculated with FIPVΔ3ABC showed temporary weight loss.

Due to advanced symptoms of FIP, one cat out of the control group had tobe euthanized at day 32, whereas the other cats in the control grouprecovered from their initial FIP symptoms. A lethality score of 25% islower than observed in the previous experiments. This is supposedly dueto the advanced age of the cats which is known to lead to reducedsusceptibility for FIPV. The three FIPVΔ3ABC+Δ7AB vaccinated cats withinitial symptoms remained ill and were euthanized between days 11 and 56(FIG. 37). Apparently, the combined deletion of the two gene clusters3ABC and 7AB which we found to reduce the fitness of FIPVΔ3ABC+Δ7AB—asjudged by its decreased in vitro growth—also affects the replicationrate of the virus in the cats, as testified by the lower levels ofneutralizing antibodies induced. Thus, the virus is over-attenuated andonly capable of partially protecting against a FIPV challenge. Fullprotection would require a higher or repeated dose.

Conclusion: Prior vaccination with FIPVΔ3ABC or FIPVΔ7AB protects catsagainst disease caused by a FIPV challenge.

II.3. Insertion and Expression of Foreign Genes:

Aim: Establish whether foreign genes can be inserted at differentpositions in the viral genome, either as an additional gene or replacingdeleted nonessential genes; and establish whether these genes areexpressed and whether they are stably maintained during in vitro passageof the virus.

II.3.a. Construction of Recombinant Viruses Carrying Reporter Genes:

Aim: Generate FIPV 79-1146 viruses with foreign gene insertions.

Procedure: A virus was constructed containing a foreign reporter gene inits genome at the position of the 3abc genes. The reporter gene, renillaluciferase (RL), was placed under the transcriptional control of the IGSproceeding gene 3a. To delete the 3abc cluster and introduce the RL geneinto the plasmid pBRDI1, combinations of primers 1244(5′-GCCATTCTCATTGATAAC-3) (SEQ ID NO:27) and 1514(5′-CTGAGTCTAGAGTAGCTAGCTAATGACTAATAAGTTTAG-3′) (SEQ ID NO:28) and of1245 (5′-GCTTCTGTTGAGTAATCACC-3) (SEQ ID NO:29) and 1513(5′-GCTAGCTACTCTAGACTCAGGCGGTTCTAAAC-3) (SEQ ID NO:30) were used togenerate fragments of 336 bp (A) and 1068 bp (B) via PCR, respectively.In primer 1513 and 1514, the underlined and bold sequences represent aNheI and XbaI restriction site, respectively. Fragments A and B werefused using the overlap between both fragments through primers 1514 and1513 and amplified using primers 1244 and 1245, using SOE-PCR, resultingin a 1384 bp fragment (C). Fragment C was cloned into the pGEM-T Easyvector (Promega), resulting in pGEM-C. The RL gene derived from pRL-null(Promega) was introduced as a NheI/XbaI fragment into NheI and XbaIdigested pGEM-C, resulting in pGEM-C+luc. Fragment C+luc was introducedas a AflII/SnaBI fragment into AflII and SnaBI-digested pBRDI1,resulting in pBRDI1Δ3ABC+luc.

After confirmation of all constructs by restriction and sequenceanalysis, recombinant viruses were generated by RNA-RNA recombinationbetween transcription vector run-off transcripts and the mFIPV genome asdescribed above. The resulting viruses were genetically confirmed byRT-PCR analysis.

Conclusion: The foreign reporter gene renilla luciferase can be placedinto the genome of the type I coronavirus FIPV. The intended recombinantviruses are viable.

II.3.b. One-Step Growth of Viruses:

Aim: Compare the growth characteristics of the viruses with wild-typevirus.

Procedure and Results: After two rounds of plaque purification, virusstocks of 2 independently obtained recombinants under II.3.a wereprepared, titrated and used for high m.o.i (m.o.i. of 8) infection ofFCWF cells after which the viral infectivities in the culture media weremonitored. The results are represented by the growth curves shown inFIG. 38. Both independently obtained recombinants grew to a 1 to 2 loglower titer as compared to that of recombinant wild-type virus.

Conclusion: The recombinant viruses with inserted expression cassettegrew normally in vitro but their yields were affected.

II.3.c. Expression of Renilla Luciferase:

Aim: Establish whether the inserted expression cassette was functional.

Procedure and Results: Confluent monolayer cultures of FCWF cells wereinfected at an m.o.i of 8 and the production of luciferase activity inthe cells was monitored over time. RL expression in cells was measuredby using the Dual-Luciferase Reporter Assay System (Promega) accordingto the manufacturer's instructions. RL was measured in relative lightunits (RLU) using a luminometer (Lumac Biocounter M2500 or TurnerDesigns Model TD-20/20).

The results are shown graphically in FIG. 39. In contrast to therecombinant wild-type virus, the two recombinant luciferase genecontaining viruses expressed high levels of luciferase activity,indicating that the renilla luciferase gene is functional. Expressionstarted as early as 2 hours post-infection. The highest expression levelwas reached at 9 hours post-infection.

Conclusion: Foreign genes can be expressed by coronaviruses by using thegenetic space created by deletion of nonessential genes.

II.4 Generation of Multivalent FIPV-Based Vaccines:

Aim: Development of multivalent vaccines based on a live attenuated FIPVstrain as a vector.

Approach: Genes (or gene fragments) from other feline and caninepathogens encoding antigens known to induce protective immunity againstthese pathogens were selected. Expression cassettes of these genes wereintroduced into the FIPV genome in combination with attenuatingdeletions of nonessential genes. The expression cassettes contain theFIPV TRS in front of (parts of) the gene to be expressed.

II.4.a. Construction of a Multivalent Feline Leukemia Virus (FeLV)Vaccine Based on FIPV Vector:

Aim: Development of FeLV vaccine based on a live attenuated FIPV strainas a vector.

Procedure: (Parts of) the protection related FeLV genes gag and env wereplaced into expression cassettes and subsequently introduced intopBRDI1Δ3ABC and pBRDI1Δ7AB, respectively. After confirmation of allconstructs by restriction and sequence analysis, recombinant viruseswere generated by RNA-RNA recombination between transcription vectorrun-off transcripts and the mFIPV genome as described above. Theresulting viruses were genetically confirmed by RT-PCR analysis.Expression of the FeLV gag and env genes was confirmed byimmunoprecipitation analysis.

Conclusion: (Parts of) the protection related FeLV genes gag and env canbe introduced into and expressed by a live attenuated FIPV strain. Theserecombinants can therefore function as a multivalent vaccine against aFeline leukemia virus and FIPV infection.

II.4.b. Construction of a Multivalent Feline Immunodeficiency Virus(FIV) Vaccine Based on an FJPV Vector:

Aim: Development of FIV vaccine based on a live attenuated FIPV strainas a vector.

Procedure: (Parts of) the protection related FIV genes gag and env wereplaced into expression cassettes and subsequently introduced intopBRDI1Δ3ABC and pBRDI1Δ7AB, respectively. After confirmation of allconstructs by restriction and sequence analysis, recombinant viruseswere generated by RNA-RNA recombination between transcription vectorrun-off transcripts and the mFIPV genome as described above. Theresulting viruses were genetically confirmed by RT-PCR analysis.Expression of the FIV gag and env genes was confirmed byimmunoprecipitation analysis.

Conclusion: (Parts of) the protection related FIV genes gag and env canbe introduced into and expressed by a live attenuated FIPV strain. Theserecombinant viruses can therefore function as a multivalent vaccineagainst a Feline immunodeficiency virus and FIPV infection.

II.4.c. Construction of a Multivalent Feline Calicivirus (FCV) VaccineBased on an FIPV Vector:

Aim: Development of FCV vaccine based on a live attenuated FIPV strainas a vector.

Procedure: (Parts of) the protection related FCV capsid gene was placedinto an expression cassette and subsequently introduced into pBRDI1Δ3ABCand pBRDI1Δ7AB, respectively. After confirmation of all constructs byrestriction and sequence analysis, recombinant viruses were generated byRNA-RNA recombination between transcription vector run-off transcriptsand the mFIPV genome as described above. The resulting viruses weregenetically confirmed by RT-PCR analysis. Expression of the FCV capsidgene was confirmed by immunoprecipitation analysis.

Conclusion: (Parts of) the protection related FCV capsid gene can beintroduced into and expressed by a live attenuated FIPV strain. Theserecombinant viruses can therefore function as a multivalent vaccineagainst a Feline calicivirus and FIPV infection.

II.4.d. Construction of a Multivalent Feline Panleucopenia Virus (FPV)Vaccine Based on an FIPV Vector:

Aim: Development of FPV vaccine based on a live attenuated FIPV strainas a vector.

Procedure: (Parts of) the protection related R3 gene, encoding the VP1and VIP2 capsid proteins was placed into an expression cassette andsubsequently introduced into pBRDI1Δ3ABC and pBRDI1Δ7AB, respectively.After confirmation of all constructs by restriction and sequenceanalysis, recombinant viruses were generated by RNA-RNA recombinationbetween transcription vector run-off transcripts and the mFIPV genome asdescribed above. The resulting viruses were genetically confirmed byRT-PCR analysis. Expression of (parts of) the R3 gene was confirmed byimmunoprecipitation analysis.

Conclusion: (Parts of) the protection related R3 gene can be introducedinto and expressed by a live attenuated FIPV strain. These recombinantviruses can therefore function as a multivalent vaccine against a Felinepanleucopenia virus and FIPV infection.

II.4.e. Construction of a Multivalent Feline Herpes Virus (FHV) VaccineBased on an FIPV Vector:

Aim: Development of FHV vaccine based on a live attenuated FIPV strainas a vector.

Procedure: (Parts of) the protection related gB, gC, gD, gE, gH, gI, gK,gL, gM, gM, ICP0, ICP1 and ICP4 feline herpes virus genes were placedinto an expression cassette and subsequently introduced into pBRDI1Δ3ABCand pBRDI1Δ7AB, respectively. After confirmation of all constructs byrestriction and sequence analysis, recombinant viruses were generated byRNA-RNA recombination between transcription vector run-off transcriptsand the mFIPV genome as described above. The resulting viruses weregenetically confirmed by RT-PCR analysis. Expression of the inserted FHPgenes was confirmed by immunoprecipitation analysis.

Conclusion: (Parts of) the protection related gB, gC, gD, gE, gH, gI,gK, gL, gM, ICP0, ICP1 and ICP4 feline herpes virus genes can beintroduced and expressed in a live attenuated FIPV strain. Theserecombinant viruses can therefore function as a multivalent vaccineagainst Feline herpes virus and FIPV.

II.4.f. Construction of a Multivalent FIPV Serotype I and II VaccineBased on an FIPV Serotype II Vector:

Aim: Development of a vaccine protecting both against serotype I andagainst II feline coronaviruses, based on a live attenuated FIPVserotype II strain as a vector.

Procedure: (Parts of) the protection related spike gene of a serotype Ifeline coronavirus, of which the region encoding the signal sequence wasdeleted, was placed into an expression cassette and subsequentlyintroduced into pBRDI1Δ3ABC and pBRDI1Δ7AB, respectively. For example:in one case, a spike gene construct was used from which the regionencoding the signal sequence had been deleted; in another case atruncated spike gene was used, from which the region encoding thetransmembrane domain+endodomain had been deleted. After confirmation ofall constructs by restriction and sequence analysis, recombinant viruseswere generated by RNA-RNA recombination between transcription vectorrun-off transcripts and the mFIPV genome as described above. Theresulting viruses were genetically confirmed by RT-PCR analysis.Expression of the inserted serotype I spike gene construct was confirmedby immunoprecipitation analysis.

Conclusion: (Parts of) the protection related spike gene of felinecoronavirus serotype I can be introduced and expressed in a liveattenuated FIPV serotype II strain and can therefore function as amultivalent vaccine against both serotype I and II feline coronaviruses.

II.5. Generation of Recombinant Viruses with Rearranged Gene Order:

Aim: Development of a FIPV vaccine in which deletion of nonessentialgenes is combined with a rearranged gene order by moving the N geneupstream of the S gene in the FIPV genome.

Procedure and Results: Capped, run-off donor transcripts weresynthesized from NotI-linearized pBRDI1Δ3ABC, pBRDI1Δ7AB andpBRDI1Δ3ABC+Δ7AB vectors in which the N gene, together with its TRS, wasinserted at a position upstream of the S. The donor transcripts wereintroduced into murine LR7 cells (80 cm² flask), that had been infectedbefore with mFIPV (m.o.i. of 0.4), by electroporation (Gene pulserelectroporation apparatus, Biorad, 2 consecutive pulses; 0.85 kV/50microF). The electroporated cells were co-cultured in a 25 cm² cultureflask with feline FCWF cells (50% confluency). After 24 hours incubationat 37° C. massive syncytia could be detected in the cell cultures.Deletion mutant viruses with rearranged genomes released into the mixedcell culture supernatant were purified by two rounds of plaquepurification on FCWF cells.

Conclusion: Deletion mutant FIPVs with rearranged gene order could begenerated. These viruses can function as live attenuated FIPV vaccines.By virtue of their rearranged gene order these viruses represent safervaccines because of their reduced ability to generate viable progeny byrecombination with viruses circulating in the field.

II.6 Generation of FIPV Based Vaccines Against Canine Pathogens:

General aim: Development of vaccines based on a live attenuated FIPVserotype II strain as a vector against canine pathogens.

Approach: Since type II feline coronaviruses express caninecoronavirus-like spike proteins (2a), such feline coronavirus vectorscan be used as vaccines in canines.

Therefore, the live attenuated FIPV vaccine that we developed can alsobe used for vaccination of canines against canine coronavirus (CCV).Furthermore, multivalent vaccines can be generated by introducingexpression cassettes of protection related genes of other caninepathogens into the FIPV genome in combination with attenuating deletionsof nonessential genes and with genome rearrangements. The expressioncassettes contain the FIPV TRS in front of (parts of) the gene to beexpressed.

II.6.a. Generation of FIPV Based Vaccine Against Canine Distemper:

Aim: Development of canine distemper vaccine based on a live attenuatedFIPV strain as a vector.

Procedure: (Parts of) the protection related H and F genes were placedinto an expression cassette and subsequently introduced into pBRDI1Δ3ABCand pBRDI1Δ7AB, respectively. After confirmation of all constructs byrestriction and sequence analysis, recombinant viruses were generated byRNA-RNA recombination between transcription vector run-off transcriptsand the mFIPV genome as described above. The resulting viruses weregenetically confirmed by RT-PCR analysis. Expression of (parts of) the Hand F genes was confirmed by immunoprecipitation analysis.

Conclusion: (Parts of) the protection related H and F genes of caninedistemper virus can be introduced into and expressed by a liveattenuated FIPV strain. These recombinant viruses can therefore functionas a vaccine against canine distemper virus and CCV infection.

II.6.b. Generation of FIPV Based Vaccine Against Canine Parvo Disease:

Aim: Development of canine parvovirus vaccine based on a live attenuatedFIPV strain as a vector.

Procedure: (Parts of) the protection related R3 gene, encoding the VP1and VIP2 capsid proteins were placed into an expression cassette andsubsequently introduced into pBRDI1Δ3ABC and pBRDI1Δ7AB, respectively.After confirmation of all constructs by restriction and sequenceanalysis, recombinant viruses were generated by RNA-RNA recombinationbetween transcription vector run-off transcripts and the mFIPV genome asdescribed above. The resulting viruses were genetically confirmed byRT-PCR analysis. Expression of (parts of) the R3 gene was confirmed byimmunoprecipitation analysis.

Conclusion: (Parts of) the protection related R3 gene of canineparvovirus can be introduced into and expressed by a live attenuatedFIPV strain. These recombinant viruses can therefore function as avaccine against canine parvovirus disease and CCV infection.

II.6.c. Generation of FIPV Based Vaccine Against Infectious CanineHepatitis:

Aim: Development of canine adenovirus serotype 1 vaccine based on a liveattenuated FIPV strain as a vector.

Procedure: (Parts of) the protection related structural protein genes ofcanine adenovirus serotype 1 were placed into an expression cassette andsubsequently introduced into pBRDI1Δ3ABC and pBRDI1Δ7AB, respectively.After confirmation of all constructs by restriction and sequenceanalysis, recombinant viruses were generated by RNA-RNA recombinationbetween transcription vector run-off transcripts and the mFIPV genome asdescribed above. The resulting viruses were genetically confirmed byRT-PCR analysis. Expression of (parts of) the protection relatedstructural protein genes of canine adenovirus serotype 1 was confirmedby immunoprecipitation analysis.

Conclusion: (Parts of) the protection related structural protein genesof canine adenovirus serotype 1 can be introduced into and expressed bya live attenuated FIPV strain. These recombinant viruses can thereforefunction as a vaccine against infectious canine hepatitis and CCVinfection.

II.6.d. Generation of FIPV Based Vaccine Against Hemorrhagic Disease ofPups:

Aim: Development of canine herpes virus 1 vaccine based on a liveattenuated FIPV strain as a vector.

Procedure: (Parts of) the protection related gB, gC, gD, gE, gH, gI, gK,gL, gM, gM, ICP0, ICP1 and ICP4 canine herpes virus genes were placedinto an expression cassette and subsequently introduced into pBRDI1Δ3ABCand pBRDI1Δ7AB, respectively. After confirmation of all constructs byrestriction and sequence analysis, recombinant viruses were generated byRNA-RNA recombination between transcription vector run-off transcriptsand the mFIPV genome as described above. The resulting viruses weregenetically confirmed by RT-PCR analysis. Expression of the insertedgenes was confirmed by immunoprecipitation analysis.

Conclusion: (Parts of) the protection related gB, gC, gD, gE, gH, gT,gK, gL, gM, ICP0, ICP1 and ICP4 canine herpes virus genes can beintroduced and expressed in a live attenuated FIPV strain. Theserecombinant viruses can therefore function as a multivalent vaccineagainst canine herpes virus 1 and CCV.

III. Transmissible Gastro-Enteritis Virus (TGEV):

III.1. Generation of a Live Attenuated Vaccine Against TGEV:

Aim: Development of a live attenuated vaccine against TGEV.

Approach: To this aim recombinant TGEV deletion mutant viruses weregenerated that lack the nonessential genes 3ab and/or 7.

Procedure: Recombinant viruses were generated as described by Almazan etal. (2000). From pBAC-TGEV^(FL), an infectious TGEV cDNA clone placedbehind a CMV promoter in pBeloBAC11, the 3ab and 7 genes were deletedvia standard cloning techniques, leaving the surrounding open readingframes and transcription regulatory sequences intact. Epithelial swinetestis (ST) cells were transfected with this pBAC-TGEV^(FL) derivativewhich lacks the 3ab and 7 genes (PBAC-TGEV^(FL)). Recombinant TGEVviruses were plaque purified and characterized by RT-PCR for the absenceof the 3ab and/or 7 genes. Large amounts of the recombinant TGEVdeletion viruses were generated by infecting ST cells.

Conclusion: Recombinant TGEV was generated that lacked the genes 3ab and7. These viruses can function as a live attenuated vaccine against TGEV.

III.2 Generation of Multivalent TGEV-Based Vaccines:

Aim: Development of multivalent vaccines based on a live attenuated TGEVas a vector.

Approach: Expression cassettes of protection related genes of porcinepathogens were introduced into the TGEV genome in combination withattenuating deletions of nonessential genes and genome rearrangements.The expression cassettes contain the TGEV TRS in front of (parts of) thegene to be expressed.

III.2.a. Construction of a Multivalent Porcine Parvovirus (PPV) VaccineBased on a TGEV Vector:

Aim: Development of a vaccine based on a live attenuated TGEV strain asa vector. Procedure: (Parts of) the protection related R3 gene, encodingthe VP1 and VP2 capsid proteins, was placed into an expression cassetteand subsequently introduced into a pBAC-TGEV^(FL) derivative which lacksgenes 3ab and 7. After confirmation of all constructs by restriction andsequence analysis, recombinant viruses were generated by transfecting STcells with this pBAC-TGEV^(FL) deletion derivative which contains (partsof) the protection related R3 gene. The resulting viruses weregenetically confirmed by RT-PCR analysis. Expression of the inserted R3gene was confirmed by immunoprecipitation analysis.

Conclusion: (Parts of) the protection related R3 gene can be introducedinto and expressed by a live attenuated TGEV strain. These recombinantviruses can therefore function as a multivalent vaccine against aPorcine Parvovirus and TGEV infection.

III.2.b. Construction of a Multivalent Swine Influenza Virus VaccineBased on a TGEV Vector:

Aim: Development of a swine influenza virus vaccine based on a liveattenuated TGEV strain as a vector.

Procedure: (Parts of) the protection related hemagglutinin (HA) andneuraminidase (NA) genes were placed into an expression cassette andsubsequently introduced into a pBAC-TGEV^(FL) derivative which lacks thegenes 3ab and 7. After confirmation of all constructs by restriction andsequence analysis, recombinant viruses were generated by transfecting STcells with this pBAC-TGEV^(FL) deletion derivative which contains (partsof) the protection related hemagglutinin (HA) and neuraminidase (NA)genes. The resulting viruses were genetically confirmed by RT-PCRanalysis. Expression of the inserted hemagglutinin (HA) andneuraminidase (NA) genes was confirmed by immunoprecipitation analysis.

Conclusion: (Parts of) the protection related hemagglutinin (HA) andneuraminidase (NA) genes can be introduced into and expressed by a liveattenuated TGEV strain. These recombinant viruses can therefore functionas a multivalent vaccine against a swine influenza virus and TGEVinfection.

III.2.c. Construction of a Multivalent African Swine Fever Virus VaccineBased on a TGEV Vector:

Aim: Development of an African swine fever virus vaccine based on a liveattenuated TGEV strain as a vector.

Procedure: (Parts of) the protection related structural protein encodinggenes were placed into an expression cassette and subsequentlyintroduced into a pBAC-TGEV^(FL) derivative which lacks the genes 3aband 7. After confirmation of all constructs by restriction and sequenceanalysis, recombinant viruses were generated by transfecting ST cellswith this pBAC-TGEV^(FL) deletion derivative which contains (parts of)the protection related structural-protein encoding genes. The resultingviruses were genetically confirmed by RT-PCR analysis. Expression of thestructural-protein encoding genes was confirmed by immunoprecipitationanalysis.

Conclusion: (Parts of) the protection related structural proteinencoding genes can be introduced into and expressed by a live attenuatedTGEV strain. These recombinant viruses can therefore function as amultivalent vaccine against an African swine fever virus and TGEVinfection.

III.2.d. Construction of a Multivalent Porcine Circovirus Type 2 VaccineBased on a TGEV Vector:

Aim: Development of a Porcine circovirus type 2 vaccine based on a liveattenuated TGEV strain as a vector.

Procedure: (Parts of) the protection related C1, C2, V1 and V2 genes ofthe Porcine circovirus type 2 were placed into an expression cassetteand subsequently introduced into a pBAC-TGEV^(FL) derivative which lacksthe genes 3ab and 7. After confirmation of all constructs by restrictionand sequence analysis, recombinant viruses were generated bytransfecting ST cells with this pBAC-TGEV^(FL) deletion derivative whichcontains (parts of) the protection related C1, C2, V1 and V2 genes ofthe Porcine circovirus type 2. The resulting viruses were geneticallyconfirmed by RT-PCR analysis. Expression of the C1, C2, V1 and V2 genesof the Porcine circovirus type 2 was confirmed by immunoprecipitationanalysis.

Conclusion: (Parts of) the protection related C1, C2, V1 and V2 genes ofthe Porcine circovirus type 2 can be introduced into and expressed by alive attenuated TGEV strain. These recombinant viruses can thereforefunction as a multivalent vaccine against a Porcine circovirus type 2,and TGEV infection.

III.2.e. Construction of a Multivalent Porcine Respiratory andReproductive Syndrome Virus Vaccine Based on a TGEV Vector:

Aim: Development of a Porcine respiratory and reproductive syndromevirus vaccine based on a live attenuated TGEV strain as a vector.

Procedure: (Parts of) the protection related ORF2 to ORF7 of the Porcinerespiratory and reproductive syndrome virus were placed into anexpression cassette and subsequently introduced into a pBAC-TGEV^(FL)derivative which lacks the genes 3ab and 7. After confirmation of allconstructs by restriction and sequence analysis, recombinant viruseswere generated by transfecting ST cells with this pBAC-TGEV^(FL)deletion derivative which contains (parts of) the protection relatedORF2 to ORF7 of the Porcine respiratory and reproductive syndrome virus.The resulting viruses were genetically confirmed by RT-PCR analysis.Expression of ORF2 to ORF7 of the Porcine respiratory and reproductivesyndrome virus was confirmed by immunoprecipitation analysis.

Conclusion: (Parts of) the protection related ORF2 to ORF7 of thePorcine respiratory and reproductive syndrome virus can be introducedinto and expressed by a live attenuated TGEV strain. These recombinantviruses can therefore function as a multivalent vaccine against aPorcine respiratory and reproductive syndrome virus and TGEV infection.

III.2.f. Construction of a Multivalent Foot-and-Mouth Disease VirusVaccine Based on a TGEV Vector:

Aim: Development of a foot-and-mouth disease virus vaccine based on alive attenuated TGEV strain as a vector.

Procedure: (Parts of) the protection related sequences encoding VP1 toVP4 of the foot-and-mouth disease virus were placed into an expressioncassette and subsequently introduced into a pBAC-TGEV^(FL) derivativewhich lacks the genes 3ab and 7. After confirmation of all constructs byrestriction and sequence analysis, recombinant viruses were generated bytransfecting ST cells with this pBAC-TGEV^(FL) deletion derivative whichcontains (parts of) the protection related sequences encoding VP1 to VP4of the foot-and-mouth disease virus. The resulting viruses weregenetically confirmed by RT-PCR analysis. Expression of the sequencesencoding VP1 to VP4 of the foot-and-mouth disease virus was confirmed byimmunoprecipitation analysis.

Conclusion: (Parts of) the protection related sequences encoding VP1 toVP4 of the foot-and-mouth disease virus can be introduced into andexpressed by a live attenuated TGEV strain. These recombinant virusescan therefore function as a multivalent vaccine against a foot-and-mouthdisease virus and TGEV infection.

III.3. Generation of Recombinant Viruses with Rearranged Gene Order:

Aim: Development of a TGEV vaccine in which deletion of nonessentialgenes is combined with a rearranged gene order by moving the N geneupstream of the S gene in the TGEV genome.

Procedure and Results: Recombinant viruses were generated as describedby Almazan et al. (2000). From pBAC-TGEV^(FL), an infectious TGEV cDNAclone placed behind a CMV promoter in pBeloBAC11, the 3ab and 7 geneswere deleted via standard cloning techniques, leaving the surroundingopen reading frames and transcription regulatory sequences intact. Inaddition the N gene was cloned to a position in the genome upstream ofthe S gene. Epithelial swine testis (ST) cells were transfected withthis pBAC-TGEV^(FL) derivative which lacks the 3ab and 7 genes and has arearranged genome. Recombinant TGEV viruses were plaque purified andcharacterized by RT-PCR for the absence of the 3ab and/or 7 genes. Largeamounts of the TGEV deletion recombinants were generated by infecting STcells.

Conclusion: Recombinant TGEV was generated that lacked the genes 3ab and7, and which has a rearranged gene order. These viruses function as alive attenuated vaccine against TGEV, as well as against other porcinepathogens when genes encoding relevant antigens are appropriatelyincorporated.

IV. Avian Infectious Bronchitis Virus (IBV):

IV.1. Generation of a Live Attenuated Vaccine Against IBV:

Aim: Development of a live attenuated vaccine against IBV.

Approach: Recombinant IBV deletion mutant viruses were generated thatlack the nonessential genes 3a/b and/or 5a/b. In order to acquirevaccine viruses that protect against multiple IBV serotypes, spike geneconstructs derived from such different viruses were incorporated.

Procedure: Recombinant viruses were generated as described by Casais etal. (2001). Infectious IBV cDNA clones were assembled in the vacciniavirus genome by using sequential in vitro ligation of cDNA fragmentsderived from pFRAG1, pFRAG2, and pFRAG3 derived plasmids, followed bydirect cloning into the genome of vaccinia virus vNotI/tk as described(1a). Genes 3a/b and 5a/b were removed from pFRAG3, by PCR mutagenesisleaving the surrounding open reading frames and transcription regulatorysequences intact, resulting in pFRAG3Δ. The S gene in pFRAG3Δ could bereplaced by S genes derived from different IBV serotypes by usingRT-PCR. Recombinant vaccinia viruses were generated by recombinationbetween the fowl pox virus HP1.441 and the vNotI/tk-IBV in vitroligation mixture. Recombinant viruses were plaque purified andcharacterized by PCR and Southern blot analysis. Finally, infectious IBVlacking genes 3a/b and/or 5a/b was generated as follows: Chick kidney(CK) cells were infected with recombinant fowl pox virus expressing T7RNA polymerase. Subsequently, cells were transfected with DNA isolatedfrom the recombinant vaccinia virus containing the IBV cDNA clonelacking genes 3a/b and 5a/b. At 3 days post-infection, the culturemedium was collected and used to infect CK cells to generate largeamounts of recombinant IBV. The recombinant viruses were geneticallyconfirmed by RT-PCR analysis.

Conclusion: Recombinant IBV was generated that lacked the genes 3a/band/or 5a/b. These viruses can function as a live attenuated vaccineagainst IBV. By replacing the S gene, recombinant IBVs could be obtainedthat function as live attenuated vaccines against different IBVserotypes.

IV.2. Generation of Multivalent IBV-Based Vaccines:

Aim: Development of multivalent vaccine based on a live attenuated IBVstrain as a vector.

Approach: Expression cassettes of protection related genes of chickenpathogens were introduced into the IBV genome in combination withattenuating deletion of nonessential genes. The expression cassettescontain the IBV TRS (CTTAACAA) (SEQ ID NO:31) in front of (parts of) thegene to be expressed.

IV.2.aI. Construction of a Multivalent Vaccine Based on an IBV Vectorthat Protects Against More than One IBV Serotype:

Aim: Development of an IBV vaccine based on a live attenuated IBV strainthat lacks nonessential genes and protects against more than oneserotype.

Procedure: (Parts of) the protection related IBV S gene from a differentIBV serotype were placed into expression cassettes, and subsequentlyintroduced into pFRAG3Δ. The largest construct contained a completeextra copy of the S gene except for the sequence encoding the signalpeptide; another construct had a carboxy-terminally truncated S genelacking the code for the transmembrane domain and endodomain. Afterconfirmation of all the constructs by restriction and sequence analysis,recombinant viruses were generated as described above. The recombinantviruses were genetically confirmed by RT-PCR analysis. The properexpression of the heterologous S gene constructs was verified byimmunoprecipitation analysis with type-specific antisera usingradiolabeled recombinant virus infected cell lysates.

Conclusion: (Parts of) the protection related IBV S gene from adifferent IBV serotype can be introduced in and expressed from a liveattenuated IBV strain. These recombinant viruses can therefore functionas multivalent vaccines to protect against infection by more than oneIBV serotype.

IV.2.aII. Construction of a Multivalent Vaccine Based on an IBV Vectorthat Protects Against Newcastle Disease:

Aim: Development of a Newcastle Disease Virus (avian paramyxovirus typeI; APMV-1) Vaccine Based on a Live Attenuated IBV Strain that LacksNonessential Genes.

Procedure: (Parts of) the protection related APMV-1 genes HN and F wereplaced in expression cassettes, and subsequently introduced intopFRAG3Δ. After confirmation of all the constructs by restriction andsequence analysis, recombinant viruses were generated as describedabove. The recombinant viruses were genetically confirmed by RT-PCRanalysis. Expression of the foreign gene was confirmed byimmunoprecipitation analysis.

Conclusion: (Parts of) the protection related APMV-1 genes HN and F canbe introduced in and expressed from a live attenuated IBV strain. Therecombinant viruses can therefore function as a multivalent vaccine toprotect against APMV-1 and IBV infections.

IV.2.b. Construction of a Multivalent Vaccine Based on an IBV Vectorthat Protects Against Avian Influenza:

Aim: Development of an Avian Influenza virus vaccine based on a liveattenuated IBV strain that lacks nonessential genes.

Procedure: (Parts of) the protection related Avian Influenza genes H andN were placed into expression cassettes, and subsequently introducedinto pFRAG3Δ. After confirmation of all the constructs by restrictionand sequence analysis, recombinant viruses were generated as describedabove. The recombinant viruses were genetically confirmed by RT-PCRanalysis. Expression of the foreign gene was confirmed byimmunoprecipitation analysis.

Conclusion: (Parts of) the protection related Avian Influenza genes Hand N can be introduced in and expressed from a live attenuated IBVstrain. These recombinant viruses can therefore function as amultivalent vaccine to protect against Avian Influenza and IBVinfections.

IV.2.c. Construction of a Multivalent Vaccine Based on an IBV Vectorthat Protects Against Chicken Anemia Virus (CAV) Disease:

Aim: Development of a CAV vaccine based on a live attenuated IBV strainthat lacks nonessential genes.

Procedure: (Parts of) the protection related CAV genes V1, V2, and V3were placed into expression cassettes, and subsequently introduced intopFRAG3A. After confirmation of all the constructs by restriction andsequence analysis, recombinant viruses were generated as describedabove. The recombinant viruses were genetically confirmed by RT-PCRanalysis. Expression of the foreign gene was confirmed byimmunoprecipitation analysis.

Conclusion: (Parts of) the protection related CAV genes V1, V2, and V3can be introduced in and expressed from a live attenuated IBV strain.These recombinant viruses can therefore function as a multivalentvaccine to protect against CAV and IBV infections.

IV.2.d. Construction of a Multivalent Vaccine Based on an IBV Vectorthat Protects Against Avian Reovirus Disease:

Aim: Development of an avian reovirus vaccine based on a live attenuatedIBV strain that lacks nonessential genes.

Procedure: (Parts of) the protection related avian reovirus genes Φ1,Φ2, and 83 were placed into expression cassettes, and subsequentlyintroduced into pFRAG3Δ. After confirmation of all the constructs byrestriction and sequence analysis, recombinant viruses were generated asdescribed above. The recombinant viruses were genetically confirmed byRT-PCR analysis. Expression of the foreign gene was confirmed byimmunoprecipitation analysis.

Conclusion: (Parts of) the protection related avian reovirus genes Φ1,Φ2, and 83 can be introduced in and expressed from a live attenuated IBVstrain. These recombinant viruses can therefore function as amultivalent vaccine to protect against avian reovirus and IBVinfections.

IV.2.e. Construction of a Multivalent Vaccine Based on an IBV Vectorthat Protects Against Infectious Bursal Disease:

Aim: Development of an Infectious Bursal Disease Virus (IBDV) vaccinebased on a live attenuated IBV strain that lacks nonessential genes.

Procedure: (Parts of) the protection related IBDV gene VP2 were placedinto expression cassettes, and subsequently introduced into pFRAG3Δ.After confirmation of all the constructs by restriction and sequenceanalysis, recombinant viruses were generated as described above. Therecombinant viruses were genetically confirmed by RT-PCR analysis.Expression of the foreign gene was confirmed by immunoprecipitationanalysis.

Conclusion: (Parts of) the protection related IBDV gene VP2 can beintroduced in and expressed from a live attenuated IBV strain. Theserecombinant viruses can therefore function as a multivalent vaccine toprotect against avian IBDV and IBV infections.

IV.2.f. Construction of a Multivalent Vaccine Based on an IBV Vectorthat Protects Against Marek's Disease:

Aim: Development of a Gallid herpes virus 2 (Marek's disease virus)vaccine based on a live attenuated IBV strain that lacks nonessentialgenes.

Procedure: (Parts of) the protection related Gallid herpes virus 2 genegB were placed into expression cassettes, and subsequently introducedinto pFRAG3Δ. After confirmation of all the constructs by restrictionand sequence analysis, recombinant viruses were generated as describedabove. The recombinant viruses were genetically confirmed by RT-PCRanalysis. Expression of the foreign gene was confirmed byimmunoprecipitation analysis.

Conclusion: (Parts of) the protection related Gallid herpes virus 2 genegB can be introduced in and expressed from a live attenuated IBV strain.These recombinant viruses can therefore function as a multivalentvaccine to protect against Gallid herpes virus 2 and IBV infections.

IV.2.g. Construction of a Multivalent Vaccine Based on an IBV Vectorthat Protects Against Infectious Laryngotracheitis:

Aim: Development of a Gallid herpes virus 1 (Infectiouslaryngotracheitis virus) vaccine based on a live attenuated IBV strainthat lacks nonessential genes.

Procedure: (Parts of) the protection related Gallid herpes virus 1 genesgB, gC, gD, gE, gH, gI, gK, gL, and gM were placed into expressioncassettes, and subsequently introduced into pFRAG3Δ. After confirmationof all the constructs by restriction and sequence analysis, recombinantviruses were generated as described above. The recombinant viruses weregenetically confirmed by RT-PCR analysis. Expression of the foreign genewas confirmed by immunoprecipitation analysis.

Conclusion: (Parts of) the protection related Gallid herpes virus 1genes can be introduced in and expressed from a live attenuated IBVstrain. These recombinant viruses can therefore function as amultivalent vaccine to protect against Gallid herpes virus 1 and IBVinfections.

IV.3. Generation of Recombinant Viruses with Rearranged Gene Order:

Aim: Development of an IBV vaccine in which deletion of nonessentialgenes is combined with a rearranged gene order by moving the N geneupstream of the S gene in the IBV genome.

Procedure: Recombinant viruses were generated as described by Casais etal. (2001). Infectious IBV cDNA clones were assembled in the vacciniavirus genome by using sequential in vitro ligation of cDNA fragmentsderived from pFRAG1, pFRAG2, and pFRAG3 derived plasmids, followed bydirect cloning into the genome of vaccinia virus vNotI/tk as described(1a). Genes 3a/b and 5a/b were removed from pFRAG3, by PCR mutagenesisleaving the surrounding open reading frames and transcription regulatorysequences intact. In addition, the N gene together with its TRS wasmoved to a position upstream of the S gene resulting inpFRAG3Δrearranged. Recombinant vaccinia viruses were generated byrecombination between the fowlpox virus HP1.441 and the vNotI/tk-IBV invitro ligation mixture. Recombinant viruses were plaque purified andcharacterized by PCR and Southern blot analysis. Finally, infectious IBVlacking genes 3a/b and 5a/b and with a rearranged gene order wasgenerated as follows: Chick kidney (CK) cells were infected withrecombinant fowl pox virus expressing T7 RNA polymerase. Subsequently,cells were transfected with DNA isolated from the recombinant vacciniavirus containing the IBV cDNA clone lacking genes 3a/b and 5a/b incombination with the rearranged gene order. At 3 days post-infection,the culture medium was collected and used to infect CK cells to generatelarge amounts of recombinant IBV. The recombinant viruses weregenetically confirmed by RT-PCR analysis.

Conclusion: Recombinant IBV was generated that lacked the genes 3a/b and5a/b in combination with a rearranged gene order. These viruses functionas a live attenuated vaccine against IBV. When combined with S geneconstructs from other IBV serotypes and/or gene constructs from otheravian pathogens, additional multivalent vaccines can be generated.

V. Human Coronavirus (HCoV) Strain 229E (HCoV-229E):

V.1. Generation of a Live Vaccine Based on Attenuated HCoV-229E:

Aim: Development of a live attenuated vaccine against HCoV-229E.

Approach: Recombinant HCoV deletion mutant viruses were generated thatlack the nonessential genes 4a/b.

Procedure: Recombinant viruses were generated essentially as describedby Thiel et al. (2001). Infectious HCoV cDNA clones were assembled inthe vaccinia virus genome. Vaccinia virus vDHCoV-vec-1 contains a 22.5kbp cDNA fragment encoding the 5′ end of the HCoV genome. This fragmentwas removed from the vaccinia genome by digestion with Bsp 120I,digested with MluI and ligated to a cDNA fragment of pMEΔ that wasobtained by digestion with MluI/EagI. pMEΔ contains a cDNA fragment thatencodes the 3′ end of the HCoV genome, but lacks gene 4a/b, withoutdisturbing the other genes or transcription regulatory sequences. pMEΔwas derived from pME by using conventional PCR mutagenesis methods. Theligation products were ligated to vNotI/tk vaccinia virus DNA.Recombinant vaccinia viruses were generated as described above.Recombinant viruses were plaque purified and characterized by PCR andSouthern blot analysis. Finally, infectious HCoV lacking genes 4a/b wasgenerated as follows: Chick kidney (CK) cells were infected withrecombinant fowl pox virus expressing T7 RNA polymerase. Subsequently,cells were transfected with DNA isolated from the recombinant vacciniavirus containing the HCoV cDNA clone lacking genes 4a/b. At 3 dayspost-infection, the culture medium was collected and used to infecthuman lung fibroblast cells (MRC-5) to generate large amounts ofrecombinant HCoV. The recombinant viruses were genetically confirmed byRT-PCR analysis.

Conclusion: Recombinant HCoV was generated that lacked the genes 4a/b.These viruses can function as a live attenuated vaccine againstHCoV-229E.

V.2. Generation of a Live Attenuated Vaccine Against HCoV Strain OC43:

Aim: Development of a live attenuated vaccine against HCoV-OC43.

Approach: Recombinant HCoV deletion mutant viruses were generated thatlack the nonessential genes 4a/b, and contain a hybrid S protein gene,which encodes the ectodomain of HCoV-OC43.

Procedure: Recombinant viruses were generated essentially as describedby Thiel et al. (2001). Infectious HCoV cDNA clones were assembled inthe vaccinia virus genome. Vaccinia virus vHCoV-vec-1 contains a 22.5kbp cDNA fragment encoding the 5′ end of the HCoV genome. This fragmentwas removed from the vaccinia genome by digestion with Bsp 120I,digested with MluI and ligated to a cDNA fragment of pMEΔ-SOC43 that wasobtained by digestion with MluI/EagI. pMEΔ-SOC43 contains a cDNAfragment that encodes the 3′end of the HCoV genome, lacks gene 4a/b, andcontains a hybrid S protein gene, without disturbing the other genes ortranscription regulatory sequences. The hybrid S protein gene containsthe region of the S gene of HCoV-OC43 that encodes the S proteinectodomain, while the remainder of the gene is derived from theHCoV-229E gene. pMEΔ-SOC43 was derived from pMEΔ by using conventional(RT-)PCR mutagenesis methods. The ligation products were ligated tovNotI/tk vaccinia virus DNA. Recombinant vaccinia viruses were generatedas described above. Recombinant viruses were plaque purified andcharacterized by PCR and Southern blot analysis. Finally, infectiousHCoV lacking genes 4a/b was generated as follows: Chick kidney (CK)cells were infected with recombinant fowl pox virus expressing T7 RNApolymerase. Subsequently, cells were transfected with DNA isolated fromthe recombinant vaccinia virus containing the HCoV cDNA clone lackinggenes 4a/b. At 3 days post-infection the culture medium was collectedand used to infect human lung fibroblast cells (MRC-5) to generate largeamounts of recombinant HCoV. The recombinant viruses were geneticallyconfirmed by RT-PCR analysis.

Conclusion: Recombinant HCoV was generated that lacked the genes 4a/b.These viruses contain the ectodomain of the S protein of HCoV-OC43 andtherefore function as a live attenuated vaccine against HCoV-OC43.

V.3. Generation of Multivalent HCoV-Based Vaccines:

Aim: Development of multivalent vaccine based on a live attenuated HCoVstrain as a vector.

Approach: Expression cassettes of protection related genes of humanpathogens were introduced into the HCoV genome in combination withattenuating deletion of nonessential genes. The expression cassettescontain the HCoV TRS (TCTCAACT) (SEQ ID NO:32) in front of (parts of)the gene to be expressed.

V.3.a. Construction of a Multivalent Vaccine Based on an HCoV Vectorthat Protects Against Respiratory Syncytial Virus (RSV):

Aim: Development of an RSV vaccine based on a live attenuated HCoVstrain that lacks nonessential genes.

Procedure: (Parts of) the protection related RSV genes HN and F wereplaced into expression cassettes, and subsequently introduced into pMEΔ.After confirmation of all the constructs by restriction and sequenceanalysis, recombinant viruses were generated as described above. Therecombinant viruses were genetically confirmed by RT-PCR analysis.Expression of the foreign gene was confirmed by immunoprecipitationanalysis.

Conclusion: (Parts of) the protection related RSV genes HN and F can beintroduced in and expressed from a live attenuated HCoV strain. Theserecombinant viruses can therefore function as a multivalent vaccine toprotect against RSV and HCoV infections.

V.3.b. Construction of a Multivalent Vaccine Based on an HCoV Vectorthat Protects Against Rotavirus:

Aim: Development of a rotavirus vaccine based on a live attenuated HCoVstrain that lacks nonessential genes.

Procedure: (Parts of) the protection related rotavirus genes VP4, VP6and VP7 were placed into expression cassettes, and subsequentlyintroduced into pMEΔ. After confirmation of all the constructs byrestriction and sequence analysis, recombinant viruses were generated asdescribed above. The recombinant viruses were genetically confirmed byRT-PCR analysis. Expression of the foreign gene was confirmed byimmunoprecipitation analysis.

Conclusion: (Parts of) the protection related rotavirus genes VP4, VP6and VP7 can be introduced in and expressed from a live attenuated HCoVstrain. These recombinant viruses can therefore function as amultivalent vaccine to protect against rotavirus and HCoV infections.

V.3.c. Construction of a Multivalent Vaccine Based on an HCoV Vectorthat Protects Against Norwalk-Like Viruses:

Aim: Development of a Norwalk-like virus vaccine based on a liveattenuated HCoV strain that lacks nonessential genes.

Procedure: (Parts of) the protection related Norwalk-like virus capsidgene was placed into expression cassettes, and subsequently introducedinto pMEΔ. After confirmation of all the constructs by restriction andsequence analysis, recombinant viruses were generated as describedabove. The recombinant viruses were genetically confirmed by RT-PCRanalysis. Expression of the foreign gene was confirmed byimmunoprecipitation analysis.

Conclusion: (Parts of) the protection related Norwalk-like virus capsidgene can be introduced in and expressed from a live attenuated HCoVstrain. This recombinant virus can therefore function as a multivalentvaccine to protect against Norwalk-like virus and HCoV infections.

V.3.d. Construction of a Multivalent Vaccine Based on an HcoV Vectorthat Protects Against Influenza Virus

Aim: Development of an influenza virus vaccine based on a liveattenuated HCoV strain that lacks nonessential genes.

Procedure: (Parts of) the protection related influenza virus genes H andN were placed into expression cassettes, and subsequently introducedinto pMEΔ. After confirmation of all the constructs by restriction andsequence analysis, recombinant viruses were generated as describedabove. The recombinant viruses were genetically confirmed by RT-PCRanalysis. Expression of the foreign gene was confirmed byimmunoprecipitation analysis.

Conclusion: (Parts of) the protection related influenza virus genes Hand N can be introduced in and expressed from a live attenuated HCoVstrain. These recombinant viruses can therefore function as amultivalent vaccine to protect against influenza virus and HCoVinfections.

V.4. Generation of Recombinant Viruses with Rearranged Gene Order:

Aim: Development of an HCoV vaccine in which deletion of nonessentialgenes is combined with a rearranged gene order by moving the N geneupstream of the S gene in the HCoV genome.

Procedure: Recombinant viruses were generated essentially as describedby Thiel et al. (2001). Infectious HCoV cDNA clones were assembled inthe vaccinia virus genome. Vaccinia virus vHCoV-vec-1 contains a 22.5kbp cDNA fragment encoding the 5′ end of the HCoV genome. This fragmentwas removed from the vaccinia virus genome by digestion with Bsp 120I,digested with MluI and ligated to a cDNA fragment of rearranged pMEΔthat was obtained by digestion with MluI/EagI. Rearranged pMEΔ containsa cDNA fragment that encodes the 3′ end of the HCoV genome, but lacksgene 4a/b, without disturbing the other genes or transcriptionregulatory sequences, and in which the N gene, together with its TRS,was positioned upstream of the S gene. Rearranged pMEΔ was derived frompME by using conventional PCR mutagenesis methods. The ligation productswere ligated to vNotI/tk vaccinia virus DNA. Recombinant vacciniaviruses were generated as described above. Recombinant viruses wereplaque purified and characterized by PCR and Southern blot analysis.Finally, infectious HCoV lacking genes 4a/b was generated as follows:Chick kidney (CK) cells were infected with recombinant fowlpox virusexpressing T7 RNA polymerase. Subsequently, cells were transfected withDNA isolated from the recombinant vaccinia virus containing the HCoVcDNA clone lacking genes 4a/b in combination with the rearranged geneorder. At 3 days post-infection, the culture medium was collected andused to infect human lung fibroblast cells (MRC-5) to generate largeamounts of recombinant HCoV. The recombinant viruses were geneticallyconfirmed by RT-PCR analysis.

Conclusion: Recombinant HCoV was generated that lacked the genes 4a/b incombination with a rearranged gene order. These viruses can function asa live attenuated vaccine against HCoV-229E. When combined with S geneconstructs from HcoV-OC43 and/or gene constructs from other humanpathogens, additional multivalent vaccines can be generated.

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1. An isolated or recombinant virus-like particle comprising nucleicacid and capable of replication in a suitable host, said isolated orrecombinant virus-like particle derived from a coronavirus wherein thecoronavirus' genes for structural proteins do not occur in the order5′-S-E-M-N-3′ and wherein the order of the coronavirus' genes forstructural proteins has been altered by gene rearrangement, wherein thegene rearrangement comprises deleting at least one gene for a structuralproteins from it's original position relative to the remaining genes forstructural proteins and inserting said deleted at least one gene for astructural protein in a different genomic location relative to theremaining genes for structural proteins.
 2. The isolated or recombinantvirus-like particle of claim 1 from which at least a fragment from anucleic acid encoding a viral gene product other than a polymerase or astructural protein N, M, E, or S, is deleted.
 3. The isolated orrecombinant virus-like particle of claim 1, wherein said isolated orrecombinant virus-like particle has been provided with at least onebiologically active protein or fragment thereof associated with thesurface of said isolated or recombinant virus-like particle other thanthe natural ectodomain of any one protein of the coronavirus from whichthe isolated or recombinant virus-like particle has been derived.
 4. Theisolated or recombinant virus-like particle of claim 2, wherein saidisolated or recombinant virus-like particle has been provided with atleast one biologically active protein or fragment thereof associatedwith the surface of said isolated or recombinant virus-like particleother than the natural ectodomain of any one protein of the coronavirusfrom which the isolated or recombinant virus-like particle has beenderived.
 5. The isolated or recombinant virus-like particle of claim 1,wherein said isolated or recombinant virus-like particle has beenprovided with a at least one functional targeting means associated withthe surface of said isolated or recombinant virus-like particle otherthan the natural spike protein of the coronavirus from which theisolated or recombinant virus-like particle has been derived.
 6. Theisolated or recombinant virus-like particle of claim 1, wherein saidisolated or recombinant virus-like particle has been provided with acoronavirus genome comprising a foreign gene or part thereof.
 7. Theisolated or recombinant virus-like particle of claim 1, which isattenuated.
 8. The isolated or recombinant virus-like particle of claim1, which is a gene delivery vehicle.
 9. The isolated or recombinantvirus-like particle of claim 1, which is an antigen or epitope deliveryvehicle.
 10. A immunogenic composition comprising: the isolated orrecombinant virus-like particle of claim 1, and a pharmaceuticallyacceptable carrier.
 11. A composition for diagnostic use comprising: theisolated or recombinant virus-like particle of claim 1 presented in amanner capable of diagnostic determination.
 12. An isolated orrecombinant virus-like particle comprising nucleic acid and capable ofreplication in a suitable host, said isolated or recombinant virus-likeparticle derived from a coronavirus wherein the coronavirus' genes forstructural proteins occur in an order selected from the group consistingof 5′-S-M-E-N-3′, 5′-E-S-M-N-3′, 5′-E-M-S-N-3′, and 5′-M-S-E-N-3′. 13.An isolated or recombinant virus-like particle comprising nucleic acidand capable of replication in a suitable host, said isolated orrecombinant virus-like particle derived from a coronavirus where thecoronavirus' genes do not occur in the order 5′-S-E-M-N-3′, and whereinonly one copy of the S, E, M, and N genes is present in therecombination virus-like particle.